Novel hydorgels and uses thereof

ABSTRACT

The present invention provides novel hydrogels and methods of making and using such hydrogels. The present invention provides hydrogels that may be formed by the self-assembly of peptides in solution. Such self-assembly may be brought about by a change in one or more characteristics of the solution. Characteristics of the solution that may be changed include pH, ionic strength, temperature, and concentration of one or more specific ions. In addition, hydrogels of the invention may be disassembled by changing one or more characteristic of the hydrogel such as pH, ionic strength, temperature, and concentration of one or more specific ions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applicationSer. No. 60/827,156, filed Sep. 27, 2006, and to U.S. provisional patentapplication Ser. No. 60/735,846 filed Nov. 14, 2005, the contents ofboth of which are specifically incorporated herein by reference.

This work was supported in part by NIH grant no. 1 P20 RR17716-01. TheU.S. Government may have certain rights to the present invention. Wethank the NIH (R01 DE016386-01) for support.

BACKGROUND OF THE INVENTION

Hydrogels are a class of materials that have significant promise for usein soft tissue and bone engineering. The general characteristic ofhydrogels that make them important materials for these applications aretheir well hydrated, porous structure. The present invention provides anew class of environmentally responsive peptide-based hydrogels thatfulfill critical material requirements not currently met with existingtechnology. Hydrogels of the invention may be designed to be compatiblewith the adhesion and proliferation of various cell types, e.g.,fibroblasts and osteoblasts, making them potential tissue engineeringscaffolds for generating connective tissue and bone. There is ademanding set of biological and material properties required ofhydrogels for use in tissue regeneration. Irrespective of ultimatetarget tissue type, a hydrogel must exhibit a general set of biologicalproperties. First, the material must be cytocompatible.Cytocompatibility, defined herein, means that the hydrogel must not becytotoxic to desired cells. Second, the material must be biocompatible.Biocompatible, defined herein, means that a scaffold does not cause asignificant immunological and inflammatory response if placed in vivofor tissue regeneration and is preferably biodegradable affordingnon-toxic species. The present invention relates to the development ofnew materials using novel self-assembly methodology and the assessmentof resultant material cytocompatibility.

Desired material properties are challenging to comprehensivelyincorporate into any one material since some desired properties areseemingly mutually exclusive. For example, the morphology of an idealhydrogel contains a high level of porosity (spanning nanoscale tomicroscale dimensions) for cell motility and nutrient/waste diffusion.Also, the hydrogel should primarily be composed of aqueous media with aslittle solid material as possible in order to allow ample volume forcell proliferation and ease of eventual scaffold biodegradation.However, despite their dilute, porous nature, these well hydratedmaterials must also be mechanically rigid. This apparent contradiction,rigidity from a dilute porous scaffold, must be inherently addressed bythe design of constituent molecular crosslinks (chemical and/orphysical) formed during the hydrogelation process. However, introducingchemical crosslinks may be biologically problematic since by-productsfrom the crosslinking chemistry may be toxic and difficult to removefrom the scaffold. Ideally, benign, biocompatible chemical or physicalcrosslinking methods should be used for either in vitro gelation foreventual incorporation in the body or direct, rapid in vivo gelationwhere formation of crosslinks are triggered by physiological stimuli(temperature, ionic strength, pH, etc). The idea of using environmentaltriggers to initiate material formation via self-assembly is beingactively pursued. For example, it has been shown that peptideself-assembly (and thus gelation) can be triggered by the release ofsalt from temperature and light sensitive liposomes (Collier, et al.,Journal of the American Chemical Society 2001, 123, 9463-9464). Anadditional design complication is that hydrogel rigidity seeminglyprecludes any viable processibility in preformed scaffolds. For example,one may wish to form a rigid tissue engineering construct in vitro butsubsequently inject it into a host for tissue regeneration. Injection isnot possible in a permanently crosslinked, rigid network.

Current hydrogel technology utilizes both naturally-derivedmacromolecules and synthetic polymers. Generally, hydrogels preparedfrom natural polymers exhibit favorable biological properties but maylack desired material properties, e.g. low sample rigidity. In contrast,synthetic polymers can be engineered for desired material properties butmay display limited cytocompatibility. A common approach to increase thecytocompatibility of synthetic polymers is to incorporate peptideepitopes, for example RGD motifs. However, incorporating these motifsinto preformed polymers in a regiospecifically controlled manner isextremely difficult to impossible. As a result, controlling the materialproperties of the polymers is problematic. For example, controlling theconcentration of an epitope displayed on the surface for cell adhesionor controlling the accessibility of the epitopes is a challenge. Inaddition, these scaffolds are structurally homogeneous (not porous) onthe microscale due to their underlying molecular network structure,which can limit cell proliferation. These systems must undergoadditional processing (e.g. freeze-thaw cycling, particulate leaching,microsphere sintering and non-woven fiber formation) in order tointroduce microscale porosity in the gel network. In short, there iscurrently no single hydrogel system that successfully incorporates allrequisite properties of an ideal tissue engineering scaffold.

An opportunity and need exists for the design of novel hydrogel scaffoldstrategies. There remains a need for rigid, porous, easily processed,cytocompatible hydrogels that can be rapidly formed in vitro or in vivo.

SUMMARY OF THE INVENTION

The present invention provides novel hydrogels. In addition, the presentinvention provides a novel process for the construction of hydrogels. Asused herein, the term hydrogel means a dilute interconnected scaffold,preferably encapsulating a large volume fraction of water andmechanically self-supporting. As used herein, the term hydrogel alsoencompasses dilute interconnected scaffolds encompassing aqueous organicmixtures and/or organic solvents (e.g., DMF, DMSO, etc.). In one aspect,the present invention provides a combination of a new process and novelpeptides to construct a smart hydrogelation system. Novel peptides (forexample, MAX1) have been designed to undergo a change in secondarystructure in response to one or more environmental signals or stimuli(e.g., changes in one or more environmental characteristics). In oneparticular aspect, peptides of the invention may be in aqueous solutionand one or more parameters of the solution may be altered in order toinduce a change in secondary structure of the peptides. In specificembodiments, one or more of pH, ionic strength, specific ionconcentration, and/or temperature of the solution may be altered and mayinduce a change in the secondary structure of the peptides. Typically,after the peptides have undergone a change in secondary structure as aresult of a change in one or more parameter of the solution, thepeptides will assemble into a higher order structure, e.g., a hydrogel.In another aspect of the invention, an environmental signal may involveelectromagnetic radiation, e.g., light. For example, a peptide of theinvention may undergo a structural change, which may be a change inprimary structure, secondary structure or both, as a result of beingsubjected to electromagnetic radiation. Typically, after being exposedto electromagnetic radiation, peptides embodying this aspect of theinvention will assume a desired secondary structure and self-assembleinto a higher order structure, e.g., a hydrogel.

In one aspect, the transition from a low viscosity aqueous solution to arigid hydrogel material (essentially an infinite change in viscosity)via a self-assembly mechanism is preferably predicated on individualpeptides folding into a desired conformation. This intramolecularfolding process can be controlled to occur only with a desiredenvironmental signal. Environmental signals include, but are not limitedto, physiological solution conditions (37 degrees Celsius, pH7.4, andhigh salt concentration). Due to robust, chemically benign gelation atphysiological conditions, these gels have major potential in the fieldof tissue engineering and wound healing.

In one particular embodiment, peptides may be designed to adopt aβ-hairpin secondary structure in response to one or more environmentalsignals. Typically, after adopting a β-hairpin structure, peptides willself-assemble into a higher order structure, for example a threedimensional network and, consequently, a hydrogel. In one aspect, theself-assembly does not take place unless side chains on the peptidemolecules are uniquely presented in the β-hairpin conformation.

Thus, in one aspect, the present invention provides a process fordesigning peptides to adopt a desired secondary structure in response toone or more environmental stimuli. Further, the present inventionprovides a process for designing a peptide that will form a higher orderstructure, e.g., a hydrogel, in response to one or more environmentalstimuli. The present invention also encompasses the higher orderstructures thus formed, e.g., hydrogels.

In some aspects, the adoption of a secondary structure and the formationof a higher order structure are linked. Thus, peptide folding andself-assembly (e.g., gelation) are linked. This aspect of the inventionallows the control of gelation. In particular, this aspect permits thecontrol of the kinetics of hydrogel formation (i.e., how fast a gel isformed). Also, control of the self-assembly process allows control ofthe physical characteristics of the hydrogel thus formed (e.g., thestiffness of the resulting gel). Peptides may be designed and/orenvironmental stimuli may be chosen such that, after application of oneor more environmental stimuli, hydrogel formation may take from about 1second to about 5 hours, from about 1 second to about 4 hours, fromabout 1 second to about 3 hours, from about 1 second to about 2 hours,from about 1 second to about 1 hour, from about 1 second to about 50minutes, from about 1 second to about 40 minutes, from about 1 second toabout 30 minutes, from about 1 second to about 20 minutes, from about 1second to about 15 minutes, from about 1 second to about 10 minutes,from about 1 second to about 5 minutes, from about 1 second to about 2minutes, from about 10 seconds to about 5 hours, from about 10 secondsto about 4 hours, from about 10 seconds to about 3 hours, from about 10seconds to about 2 hours, from about 10 seconds to about 1 hour, fromabout 10 seconds to about 50 minutes, from about 10 seconds to about 40minutes, from about 10 seconds to about 30 minutes, from about 10seconds to about 20 minutes, from about 10 seconds to about 15 minutes,from about 10 seconds to about 10 minutes, from about 10 seconds toabout 5 minutes, from about 10 seconds to about 2 minutes, from about 30seconds to about 5 hours, from about 30 seconds to about 4 hours, fromabout 30 seconds to about 3 hours, from about 30 seconds to about 2hours, from about 30 seconds to about 1 hour, from about 30 seconds toabout 50 minutes, from about 30 seconds to about 40 minutes, from about30 seconds to about 30 minutes, from about 30 seconds to about 20minutes, from about 30 seconds to about 15 minutes, from about 30seconds to about 10 minutes, from about 30 seconds to about 5 minutes,from about 30 seconds to about 2 minutes, from about 60 seconds to about5 hours, from about 60 seconds to about 4 hours, from about 60 secondsto about 3 hours, from about 60 seconds to about 2 hours, from about 60seconds to about 1 hour, from about 60 seconds to about 50 minutes, fromabout 60 seconds to about 40 minutes, from about 60 seconds to about 30minutes, from about 60 seconds to about 20 minutes, from about 60seconds to about −15 minutes, from about 60 seconds to about 10 minutes,from about 60 seconds to about 5 minutes, or from about 60 seconds toabout 2 minutes.

Hydrogels formed according to the invention may have varying amounts ofsolid material. For example, hydrogels may be formed comprising apercent by weight of peptide of from about 0.1% to about 10.0%, fromabout 0.1% to about 9.0%, from about 0.1% to about 8.0%, from about 0.1%to about 7.0%, from about 0.1% to about 6.0%, from about 0.1% to about5.0%, from about 0.1% to about 4.0%, from about 0.1% to about 3.0%, fromabout 0.1% to about 2.0%, from about 0.1% to about 1.0%, from about 0.1%to about 0.75%, from about 0.1% to about 0.5%, from about 0.1% to about0.25%, from about 0.25% to about 10.0%, from about 0.25% to about 9.0%,from about 0.25% to about 8.0%, from about 0.25% to about 7.0%, fromabout 0.25% to about 6.0%, from about 0.25% to about 5.0%, from about0.25% to about 4.0%, from about 0.25% to about 3.0%, from about 0.25% toabout 2.0%, from about 0.25% to about 1.0%, from about 0.25% to about0.75%, from about 0.25% to about 0.5%, from about 0.5% to about 10.0%,from about 0.5% to about 9.0%, from about 0.5% to about 8.0%, from about0.5% to about 7.0%, from about 0.5% to about 6.0%, from about 0.5% toabout 5.0%, from about 0.5% to about 4.0%, from about 0.5% to about3.0%, from about 0.5% to about 2.0%, from about 0.5% to about 1.0%, fromabout 0.5% to about 0.75%, from about 1.0% to about 10.0%, from about1.0% to about 9.0%, from about 1.0% to about 8.0%, from about 1.0% toabout 7.0%, from about 1.0% to about 6.0%, from about 1.0% to about5.0%, from about 1.0% to about 4.0%, from about 1.0% to about 3.0%, fromabout 1.0% to about 2.0%, or from about 1.0% to about 1.5%.

In one aspect, the amount by weight of peptide and the kinetics ofgelation may be varied to produce a hydrogel having a desired modulus(stiffness). Hydrogels of the invention may have a modulus from about 1Pascal (Pa) to about 100,000 Pa, from about 1 Pa to about 50,000 Pa,from about 1 Pa to about 25,000 Pa, from about 1 Pa to about 10,000 Pa,from about 1 Pa to about 7,500 Pa, from about 1 Pa to about 5,000 Pa,from about 1 Pa to about 2,500 Pa, from about 1 Pa to about 2,000 Pa,from about 1 Pa to about 1,500 Pa, from about 1 Pa to about 1,000 Pa,from about 1 Pa to about 500 Pa, from about 1 Pa to about 250 Pa, fromabout 1 Pa to about 100 Pa, from about 100 Pa to about 100,000 Pa, fromabout 100 Pa to about 50,000 Pa, from about 100 Pa to about 25,000 Pa,from about 100 Pa to about 10,000 Pa, from about 100 Pa to about 7,500Pa, from about 100 Pa to about 5,000 Pa, from about 100 Pa to about2,500 Pa, from about 100 Pa to about 2,000 Pa, from about 100 Pa toabout 1,500 Pa, from about 100 Pa to about 1,000 Pa, from about 100 Pato about 500 Pa, or from about 100 Pa to about 250 Pa.

In one aspect of the invention, the hydrogels formed may be processed.For example, hydrogels of the invention may be injected into an animal(e.g., mammal). Since hydrogels of the invention self-assemble, one caneasily process the stiff gel (e.g. inject through a syringe) while itimmediately reassembles/stiffens after the cessation of processing. In arelated aspect, hydrogels may be formed physically via self-assemblyfrom a low viscosity solution, thus, hydrogels may be produced inrestrictive geometries, in vitro or in vivo.

In some preferred embodiments, peptide-based hydrogels of the inventionare completely noncytotoxic and may also promote the adhesion andproliferation of common mammalian cells (e.g. stem cells, fibroblasts,osteoblasts). Thus, hydrogels of the invention may be used in theculture of cells. Cell cultures can be encapsulated in three dimensionsdue to self-assembly mechanism thus allowing 3-D cell attachment andproliferation. In some embodiments, hydrogels may be used asthree-dimensional supports to grow/maintain cells lines that have beenengineered to produce therapeutics such as pharmaceutical compounds,peptides, proteins, antibodies and the like. Continuous flow of mediathrough a bioreactor containing hydrogel and cells affords rapidisolation of compounds and a means of continual cell proliferation.

In one specific embodiment, hydrogels of the invention may be used forboth two-dimensional and three-dimensional stem cell culturing. Stemcells include embryonic and tissue-derived as well as tissue-derivedcells cultured to display embyryonic phenotype. Hydrogels of theinvention may be used for the delivery of stem cells to targeted tissuetypes via injection for tissue engineering applications. In a relatedembodiment, the present invention provides a method of encapsulatingcells (e.g., stem cells) in a hydrogel by providing a solutioncomprising peptides and a solution comprising cells and combining thesolutions such that a characteristic of the solution comprising peptidesis altered such that a hydrogel is formed. The characteristic adjustedmay be selected from a group consisting of pH, ionic strength, andspecific ion concentration. In a particular embodiment, thecharacteristic adjusted may be ionic strength. In another embodiment,the characteristic adjusted may be Ca²⁺ ion concentration. Any type ofcell may be encapsulated, for example, animal cells, mammalian cells,human cells, stem cells, osteoblasts, ad/or fibroblasts. A cellencapsulated according to the present invention may be a recombinantcell, i.e., may contain exogenous nucleic acid material. Typically,cells encapsulated according to the invention may express one or moremolecules of interest, e.g., protein of interest such as an antibody. Ina particular embodiment, cells encapsulated according to the inventionmay be stem cells, e.g., pluripotent stem cells. In one embodiment,cells encapsulated according to the invention may be human stem cells.

In some aspects, hydrogels prepared from peptides of the invention(e.g., MAX-1 and related hairpin peptides) exhibit antimicrobialbehavior against gram positive and gram negative bacteria. Therefore,hydrogels of the invention may be antimicrobial in a clinical setting.This characteristic of hydrogels of the invention will make them usefulin situations in which the hydrogel is to be placed inside a livinganimal (e.g., a mammal such as a human) as well in methods of culturingcells. In a particular embodiment, hydrogels of the invention may beused for tissue engineering. For example, a desired quantity of one ormore types of cell may be placed in solution with one or more peptidesof the invention. The cell containing solution may be caused to form ahydrogel in which the cells may be dispersed throughout the hydrogel.The cell containing hydrogel may then be used as tissue, for example, toreplace a damaged tissue. The antimicrobial character of the hydrogelsof the invention will help to prevent infection when introduced into ananimal. In some embodiments, the hydrogels of the invention may beconstructed to undergo reversible gelation, i.e., to form a hydrogelunder one set of conditions and then go back into solution under otherconditions. This may be used in tissue engineering application such thatcells may introduced in a hydrogel scaffolding that eventually dissolvesleaving the cells in place. Antimicrobial gels will also be useful inwound healing applications. Hydrogels for use in wound healingapplications may comprise therapeutic agents in addition to thehydrogel. For example, a solution of peptides that undergo hydrogelformation when placed in physiological conditions (i.e., in contact witha wound) may comprise agents to promote coagulation, analgesics and/orother therapeutic agents.

In some aspects, hydrogels of the invention may be readilyfunctionalized with further biochemistry (e.g. growth factor or celladhesion peptide epitopes) for further optimization. Thus, peptides ofthe invention may comprise additional components, which may be peptides,that give the hydrogels of the invention varying characteristics.

Hydrogels of the invention may be used in microfluidic devices asenvironmentally responsive barrier materials. For example, the channelsof a microfluidic devices can flooded with an aqueous solution ofpeptide. Hydrogelation can initiated with spatial resolution to installchannel barriers at desired locations. These barriers or dams can act assensing devices. When solution passes over them which causes hydrogeldissolution (dam destruction), nascent channels are opened allowingfluid to flow to detectors, providing the means of detection of analyte.The dissolution of hydrogel barriers can also be used to initiate andfacilitate mixing of reacting components for desired chemicaltransformations and reactions.

The hydrogels of the invention may be used as sensors for the detectionof one or more analyte of interest. For example, a solution of peptidesof the invention may be contacted with a sample that may contain theanalyte. In some instances, presence of the analyte may induce gelationof the solution. For example, the analyte of interest may be a metal ionand contact of the solution of peptides with the metal ion may result ingelation of the solution. In other instances, a hydrogel of theinvention may be formed and then contacted with a sample that maycontain the analyte of interest. The presence of the analyte may resultsolubilizing the hydrogel. The detection of the formation or dissolutionof hydrogel may be accomplished using standard techniques well known toone of ordinary skill in the art, for example, optical techniques.

In some embodiments, hydrogels of the invention may be used to preparematrices for separation of molecules of interest (e.g., biomolecules,proteins, DNA, RNA, etc.). Peptides may be designed to havecharacteristics useful for separation of the molecules of interest. Forexample, moieties capable of specific interactions with a molecule ofinterest may be designed into peptides that are used to make a hydrogel.Suitable moieties capable of specific interaction include, but are notlimited to, epitopes, ligands, specific small molecules, nucleic acidsequences, and the like. Hydrogels of this type may be used in either apositive selection (i.e., binding the molecule of interest) or negativeselection (i.e., binding contaminants) mode. For use in embodiments ofthis type, it may be desirable to control the nanoporous and miroporousmorphology of the hydrogels in order to purify the desired molecule. Forexample, the size of the pores in the hydrogels of the invention may becontrolled by varying the amount by weight of peptide used, gelationconditions, and/or the peptide structure to optimize purification of amolecule of interest.

The hydrogels of the invention may be used to improve the toleranceand/or adhesion of materials placed in a living organism. For example,material to be implanted in a living organism (e.g., prosthetics, pacemakers, supports, etc) may be first coated with a hydrogel of theinvention. Such hydrogels may be made of peptides having one or moremoieties that promote the adhesion of the tissues of the organism to theimplanted device. Such moieties may include adhesion epitopes and thelike. Hydrogels may also include immune modulating (e.g., suppressing orstimulating) moieties such as small molecules or epitopes.

In another embodiment, hydrogels of the invention may be used forharmful metal ion remediation from aqueous solutions. Peptides may bedesigned that contain functionalities that bind to the harmful metalion. The peptides may be introduced into a solution comprising theharmful metal ion and then the gelation of the peptides may be induced.The harmful metals may be trapped inside the hydrogel and the hydrogelmay be separated from the rest of the solution by any suitabletechnique, for example, by filtration. Optionally, the hydrogel may bedissolved and the harmful metal ion may be isolated.

In some aspects, the present invention provides a method of making ahydrogel. Such a method may entail providing a solution comprisingpeptides and altering one or more characteristics of the solution,wherein a hydrogel is formed. The characteristic altered may be anycharacteristic that results in formation of a hydrogel upon itsalteration. Suitable examples include, but are not limited to, ionicstrength, temperature, concentration of a specific ion, and pH. In someembodiments, altering one or more characteristic of the solution maycomprise contacting the solution with electromagnetic radiation. Inparticular embodiments, the character altered may be the pH of thesolution. In some embodiments, altering one or more characteristic ofthe solution results in a salt concentration of from about 20 mM toabout 400 mM. Any salt may be used, for example, KCl, NaCl, MgCl₂, KF,MgSO₄, etc. In one embodiment, the salt may be NaCl. In someembodiments, the solution may have a desired pH, for example, a pH ofless than 9, a pH of from about 6.0 to about 8.5, a pH of from about 7.0to about 8.0, or a pH of about 7.4, which may stay the same or bechanged upon formation of the hydrogel.

In some aspects, the present invention provides a hydrogel. Suchhydrogels may comprise peptides and from about 20 mM to about 400 mMsalt. As discussed above, any salt may be used, for example, NaCl. Anypeptide capable of forming a hydrogel may be used, for example, MAX1.

In one aspect, the present invention provides a method of making ahydrogel. Such a method may comprise injecting a solution comprisingpeptides into an animal, wherein the solution forms a hydrogel insidethe animal. Any animal may be used, for example, mammals includinghumans. A solution for use in this aspect of the invention may compriseany number of components. In some embodiments, the solution may compriseone or more therapeutic agents. Any therapeutic agent known to thoseskilled in the art may be used. In particular embodiments, the solutionsmay comprise one or more therapeutic agent selected from a groupconsisting of small molecules, peptides, proteins, and cells.

In another aspect of the invention, the invention provides a method ofdelivering a therapeutic agent to an animal in need thereof. Such amethod may comprise administering a solution comprising the therapeuticagent and one or more peptides to the animal, wherein the solution formsa hydrogel inside the animal. Such a method may be practiced on any typeof animal including mammals such as humans. Any type of therapeuticagent known to those skilled in the art may be used, for example, smallmolecules, peptides, proteins, and cells. One of ordinary skill in theart will appreciate that a therapeutic agent is any agent that resultsin the prevention and/or amelioration of any undesirable condition.

In another aspect, the present invention provides a method of deliveringa therapeutic agent to an animal in need thereof, comprisingadministering a hydrogel comprising the therapeutic agent and one ormore peptides to the animal. Such a method may be practiced on any typeof animal including mammals such as humans. Any type of therapeuticagent known to those skilled in the art may be used, for example, smallmolecules, peptides, proteins, and cells.

In another aspect, the present invention provides a method of treating awound in an animal. Such a method may comprise contacting the wound witha solution comprising a peptide, wherein the solution forms a hydrogel.Solutions for use in this aspect of the invention may further compriseone or more therapeutic agents. Methods of this type may be practiced onany type of animal, for example, mammals including humans. Any type oftherapeutic agent known to those skilled in the art may be used, forexample, small molecules, peptides, proteins, and cells.

In another aspect, the present invention provides a method of growingcells. Such methods may comprise forming a hydrogel comprising cells andmaintaining the cells under conditions suitable for cell viability.Hydrogels for use in this aspect of the invention typically comprisepeptides. Hydrogels may be formed, for example, by adjusting one or morecharacteristic of a solution comprising peptides. The characteristicadjusted may be one or more of pH, ionic strength, and specific ionconcentration. In one particular embodiment, the characteristic adjustedis ionic strength. In another particular embodiments, the characteristicadjusted is Ca²⁺ ion concentration. Any type of cell may be grown usingmethods of the invention, for example, animal cells such as mammaliancells including human cells. In some particular embodiments, the cellsmay be stem cells, osteoblasts or fibroblasts. Cells to be grown usingmethods of the invention may be recombinant cells, i.e., may contain oneor more exogenous nucleic acid molecules. Such nucleic acid moleculesmay be incorporated into the genome of the cell and/or may be maintainedextra-chromosomally. Cells to be grown using methods of the inventionmay express a protein of interest. Examples of proteins of interestinclude, but are not limited to, antibodies.

In another aspect, the present invention provides a sensor comprising ahydrogel. Hydrogels for use in sensors of the invention may have one ormore characteristic that is altered when the hydrogel is contacted withan analyte of interest. An analyte of interest is any material desiredto be detected. In some embodiments, the characteristic of the hydrogelaltered in response to the analyte is stiffness. In other embodiments,the characteristic altered is an optical property. Optical propertiesinclude, but are not limited to, absorbance, ellipticity, lightscattering characteristics and the like.

In another aspect, the present invention provides a method of detectingenvironmental conditions. Such methods may entail contacting a sensorcomprising a hydrogel with a sample representative of the environmentalconditions and determining one or more characteristic of the hydrogel.Typically, in methods of this type, one or more characteristic of thehydrogel is altered when the hydrogel is contacted with an analyte ofinterest. Any characteristic of the hydrogel may be altered, forexample, stiffness and/or an optical property. Methods of this type mayalso include comparing the characteristic of the hydrogel to the samecharacteristic of the hydrogel determined at a different time.

In another aspect, the present invention provides methods of purifying amolecule of interest. Such methods may include contacting a solutioncomprising the molecule of interest and one or more contaminants with ahydrogel under conditions causing the molecule of interest to beretained by the hydrogel and recovering the molecule of interest fromthe hydrogel. Typically, in methods of this type, at least onecontaminant is not retained by the hydrogel or is retained to a lesserdegree than the molecule of interest. Molecules of interest may be anymolecule known to one of ordinary skill in the art. Examples ofmolecules of interest include proteins, nucleic acid molecules, smallmolecules and the like. In one particular embodiment, the molecule ofinterest may be an antibody. A molecule of interest may be a therapeuticagent or a component part of a therapeutic agent. A component part of atherapeutic agent is a material that may be modified to become atherapeutic agent. One example of a component part of a therapeuticagent is an antibody that may be covalently modified with a cytotoxiccompound to become a therapeutic agent.

In another aspect, the present invention provides a method of purifyinga molecule of interest including contacting a solution comprising themolecule of interest and one or more contaminants with a hydrogel underconditions causing at least one contaminant to be retained by thehydrogel and recovering the molecule of interest. Typically, in methodsof this type, at least one contaminant is retained by the hydrogel or isretained to a greater degree than the molecule of interest. Molecules ofinterest may be any molecule known to one of ordinary skill in the art.Examples of molecules of interest include proteins, nucleic acidmolecules, small molecules and the like. In one particular embodiment,the molecule of interest may be an antibody. A molecule of interest maybe a therapeutic agent or a component part of a therapeutic agent. Acomponent part of a therapeutic agent is a material that may be modifiedto become a therapeutic agent. One example of a component part of atherapeutic agent is an antibody that may be covalently modified with acytotoxic compound to become a therapeutic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of the self-assembly of peptidesto form a hydrogel according to the present invention. Unfolded peptidesare induced to individually fold into a conformation amenable toself-assembly with other like-folded peptides. The folding process iscaused by exposure to desired stimuli and the self-assembly processresults in an interconnected, fibrillar, hydrogel network.

FIG. 2 shows a schematic representation of some of the factors that canbe varied in peptides used in preparing hydrogels having desiredcharacteristics according to the present invention. The specificmolecular factors that can be changed include:

FIG. 2A-describes manipulations to influence intramolecular folding andinclude: 1) electrostatic interactions between the primary aminechemical functionality of the lysine residues of individual peptides, 2)hydrophobic, van der Waals interactions between peptide backbone arms ofindividual peptides, 3) hydrogen bonding between arms of an individualpeptide backbone, 4) turn propensity of individual peptides (i.e. the“stength” of a peptide to fold relative to a particular stimulus).

FIG. 2B-describes manipulations to influence intermolecularself-assembly and include: 5) hydrophobic, van der Waals interactionsbetween neighboring folded peptide valine-rich faces, 6) hydrophobic,van der Waals interactions between peptide backbone arms of neighboringpeptides, 7) hydrogen bonding between peptide backbone arms ofneighboring peptides.

FIG. 3A shows the structure of a peptide for use in the presentinvention. FIG. 3B shows a schematic representation of the reversibleformation of a hydrogel according to the invention. Specifically, thepeptide MAX1 is unstructured unfolded under acidic or neutral pHsolution conditions. When exposed to basic solution conditions the transprolyl amide bond in the MAX1 turn sequence forces the arms ofindividual peptides into a parallel conformation stabilized by theinteractions listed in FIG. 2A. Self-assembly consequently occurs due tointeractions described in FIG. 2B. If the resultant hydrogel is exposedto acidic pH conditions the peptides unfold and disassemble back into adilute, low viscosity aqueous solution.

FIG. 4 shows experimental results obtained from a hydrogel preparedaccording to the invention. FIG. 4A shows the time-dependent far UV-CDspectra of a 150 μM MAX1 solution (pH 9.0, 125 mM Borate, 10 mM NaCl).FIG. 4B shows [θ]₂₁₆ monitored as a function of time for 150 and 250 μMMAX1 solutions under identical conditions. FIG. 4C shows [θ]₂₁₆monitored as a function of time and pH for a 300 μM solution of MAX1showing that the random coil-sheet folding/self-assembly equilibria arereversible. All experiments performed at 20° C.

FIG. 5 shows an FTIR; 1 wt % MAX1 at pH 5.5 (trace a) and pH 9.0, aftergelation (trace b). The large shift in the amide 1 stretch from 1644 in(a) to 1615 in (b) strongly indicates the growth of beta-sheetstructure.

FIG. 6 shows rheology data on 2 wt % MAX1 hydrogel at pH 9. FIG. 6Ashows the quick hydrogelation monitored at 6 Hz, 1% strain as a functionof time via increase in storage (G′, solid symbols) and loss (G″, opensymbols) shear moduli. FIG. 6B shows frequency sweep data. Theinsensitivity of the moduli to frequency indicates a stronglycrosslinked, rigid hydrogel due to the peptide folding andself-assembly. FIG. 6C shows rate sweep data (viscosity, open symbols;stress, closed symbols) indicative of shear thinning material that iseasy to post-assembly process (e.g. inject through a syringe). FIG. 6Dshows restoration of gel moduli as a function of time after thecessation of strain treatment (1000% strain at 6 Hz for 180 s). Symbolsare as defined for FIG. 6A. Immediately after hydrogel destruction vialarge strain the gel immediately solidifies back to ˜75% of its formerrigidity and quickly (˜10-20 minutes) regains its prestrain rigidity.

FIG. 7 shows results obtained with laser scanning confocal microscopy(LSCM) and cryo transmission electron microscopy (cryoTEM). FIG. 7Ashows LSCM of hydrogel microstructure. Green regions are fluorescentlystained self-assembled peptide and black regions are water filled poresand channels. Space bar is =20 μm. FIG. 7B shows cryoTEM ofself-assembled nanostructure. Dark structures are self-assembled peptidescaffold while lighter gray areas are composed of vitrified water. Spacebar is equal to 200 nm.

FIG. 8 shows combined USANS/SANS plot of 1 wt % MAX1 hydrogel. (Inset isan enlargement of the log(intensity) from 0.02<q<0.08 with a nonlinear,least-squares fit of −1.1). The −4 slope at low q is indicative of amicroporous structure as can be seen microscopically in FIG. 7A. The −1slope in the inset is indicative of a local rod-like structure as can beseen microscopically in FIG. 7B.

FIG. 9A shows temperature dependent CD of a 150 μM solution of MAX1 (125mM Borate, 10 mM NaCl, pH 9). At low temperature MAX1 is unfolded (andunassembled). At high temperature MAX1 folds into beta-sheet (andconsequently self-assembles into a hydrogel). FIG. 9B shows temperaturedependence of [θ]₂₁₈ for MAX1, 2 and 3 under identical conditions whereMAX1=VKVKVKVKV^(D)PPTKVKVKVKV-NH₂, MAX2=VKVKVKVKV^(D)PPTKVKTKVKV-NH₂ andMAX3=VKVKVKTKV^(D)PPTKVKTKVKV-NH₂. The relative hydrophobicity isMAX1>MAX2>MAX3 due to the isostructural but less hydrophobicsubstitutions of threonine for valine at the underlined positions. Themost hydrophobic folds at the lowest temperature while the leasthydrophobic folds at the highest temperature. This is a directmanifestations of the molecular parameter manipulations described inFIG. 2. Also shown are the calculated free energy of transfer of eachcorresponding unfolded peptide having an overall +8 charge state fromoctanol into water at 25° C. MAX1, the most hydrophobic has the highestfree energy of transfer equal to 7.60 kcal/mol. MAX2 has a calculatedtransfer free energy of 6.88 kcal/mol, while the least hydrophobic MAX3has the lowest energy of transfer of 6.45 kcal/mol.

FIG. 10A shows reversible temperature dependent CD of a 150 μM solutionof MAX3 (125 mM Borate, 10 mM NaCl, pH 9). ∘ is MAX3 at 5° C. showingcompletely unfoled character. □ is MAX 3 after heating to 80° C. andundergoing intramolecular folding. ♦ is MAX3 after cooling back to 5° C.showing completely unfoled character. ▴ is MAX 3 after reheating to 80°C. and undergoing intramolecular folding. FIG. 10B shows temperaturedependency of the storage modulus (G′) for a 2 wt % aqueous preparationof MAX3 under identical conditions; data was collected at the indicatedtemperatures for 20 min. time intervals allotting time for approximateinstrumental/sample equilibrium between intervals. The rheology clearlyshows reversible self-assembly and consequent solid hydrogel rigiditywith temperature.

FIG. 11 shows the structure of various peptides that may be used in thepractice of the instant invention. FIG. 11A shows the structure of MAX1,in which valines incorporated at H-bonding positions within each hairpinproject their side chains outward making intermolecular lateralhydrophobic contacts (as described in FIG. 2) possible which help driveself-assembly. FIG. 11B shows the structure of MAX4, in which valinesare now incorporated at non H-bonding positions and project their sidechains inward; valine-derived intermolecular interactions are lesslikely and self-assembly is not as favorable.

FIG. 12 shows the results of a rheology study. Rate of storage modulusincrease for 1 wt % preparations of MAX1 and MAX4 at 40° C. (pH9.0, 125mM Borate, 10 mM NaCl); frequency=6 rad/sec. MAX1 clearly assembles morequickly than MAX4 due to increased intermolecular, lateral hydrophobiccontact.

FIG. 13 shows a TEM micrograph of MAX9 fibrils negatively stained withuranyl acetate. This stiff, irreversible fibrils are due to thenonfolding nature of MAX9 and lead to no hydrogelation.

FIG. 14A shows the salt dependent CD of 150 μM MAX1 at pH 7 (20 mMTris). At 20 mM KF salt the CD indicates a completely unfoldedconformation ●. At 150 mM KF the CD indicates strong beta-sheetconformation formation ▪. FIG. 14B shows frequency sweep data for MAX1hydrogel formed at pH 7 with serum free growth media. The frequencyindependence of the moduli (G′=●, G″=▪) clearly indicate a heavilycrosslinked, rigid hydrogel material.

FIG. 15 shows the storage modulus of 2 wt % MAX1 versus time atdifferent temperatures (pH 7.4, 1% strain, 6 rad/s). ●=20°, ▴=37° C.,and ♦=60° C. The higher the temperature, the more quickly the peptidefolds and assembles and the more rigid the resultant hydrogel network.

FIG. 16 shows light microscopy results. FIG. 16A shows 10⁴ fibroblastsin DMEM with 10% calf serum plated on 2 wt % Max1 gel, t=4.5 hours. FIG.16B shows the results from 16A at t=72 h. FIG. 16C shows the control,10⁴ fibroblasts in DMEM with 10% calf serum plated on polystyrene, t=4h. FIG. 16D shows the control at t=72 h. FIG. 16E shows 10⁴ fibroblastsin DMEM without serum plated on 2 wt % Max1 gel, t=4.5 hours. FIG. 16Fshows the same material as FIG. 16E with calf serum added to the sampleat 4 h and incubated for 72 h.

FIG. 17 shows the results of a proliferation assay of NIH 3T3fibroblasts on MAX1 hydrogels versus cell culture polystyrene at varyinginitial cell seeding densities. The clear bars indicate cellproliferation on tissue culture polystyrene control while the shadedbars are the cell proliferation on 2 wt. % MAX1 hydrogel. Theproliferation on the self-assembled hydrogel was higher than the controlat all cell seeding densities.

FIG. 18 is an LSCM image showing that fibroblasts are impregnatedthroughout the MAX1 hydrogel. Z-stack image viewed perpendicular toZ-axis. Bar=100 μm Therefore, due to the self-assembly mechanism ofhydrogel formation, cells can readily be encapsulated in threedimensions.

FIG. 19 shows a schematic representation of a peptide for use in theinvention indicating locations in the peptide that may be modified. Cellbinding epitopes, such as the sequence arginine, glycine, aspartic acid(RGD) can be incorporated at 1) one or both of the peptide ends, 2) inthe middle of one or both of the peptide strands, 3) at one or both ofthe turn flanking positions, or 4) any lysine side chain via covalentattachment.

FIG. 20 shows examples of triggers that may be used in connection withthe present invention. 330-360 nm wavelength light can be used as anintramolecular folding (and, thus, hydrogelation) trigger byincorporation of chemistries shown in the figure at Val(16) (schemes 1and 2). Calcium II binding can be used as an intramolecular folding(and, thus, hydrogelation) trigger by incorporation of chemistries shownin schemes 3 and 4 into the turn sequence.

FIG. 21A shows a CD spectra of 2 wt % Max1, pH 7.4 solution with 0 mM(∘) and 150 mM (●) KF at 20° C. showing clear beta-sheet formation withsalt. FIG. 21B shows an FTIR spectrum of 2 wt % Max1 solution, pH7.4with 0 and 400 mM NaCl again showing clear beta-sheet formation withsalt addition. FIG. 21C shows a WAXS spectrum of 3 wt % Max1 solution,pH 7 with 150 mM NaCl. The peak represents a spacing of 4.7 Å, theintermolecular spacing of peptides in a beta-sheet conformation.

FIG. 22A shows a dynamic frequency sweep (5% strain) of 2 wt % Max1,pH7.4 solution with 20 mM (G′: ●, G″:∘), 150 mM (G′: ▴, G″:Δ), and 400mM (G′:▪, G″:□) NaCl at 20° C. The higher the salt concentrationstimulus, the more rigid the hydrogel network. FIG. 22B shows dynamictime sweep (1% strain, 6 rad/s) of 2 wt % Max1 solution with 20 mM (G′:●), 150 mM (G′:▴) and 400 mM (G′:▪) NaCl at 20° C. The higher the saltconcentration, the more quick the folding and consequent self-assembly,and the more rigid the ultimate hydrogel material.

FIG. 23 shows negatively stained (Uranyl acetate) TEM images ofself-assembled structure of hydrogels. FIG. 23A shows a denselyinterconnected network of 2 wt % Max1, pH7.4, 400 mM NaCl solution. FIG.23B shows fibrillar assemblies of a diluted hydrogel (finalconcentration after dilution: ˜0.1 wt %). The nanostructure is inagreement with the network structure schematically shown in FIG. 1 andFIG. 24.

FIG. 24 shows the proposed structure of self-assembled Max1 β-Hairpinmolecules in a fibril. The stand axis of the molecule is 32 Å and thecross-section thickness is 20 Å. The long axis of the structure showsthe hydrogen bonding and fibril growth direction.

FIG. 25A shows dynamic time sweep data (1% strain, 6 rad/s) of 2 wt %Max1, pH7.4 solution with 150 mM NaCl at 20° C. (G′:∘) and 37° C. (G′:Δ). The higher the temperature, the more quick the assembly and the morerigid the ultimate material. FIG. 25B shows frequency sweep data (5%strain) of hydrogel at 37° C. after 2.5 hours of gelation (G′: ●, G″:∘).FIG. 25C shows time-dependent mean molar ellipticity at 218 nm ([θ]₂₁₈)of 2.0 wt % Max1, pH 7.4 with 150 mM NaCl at 20° C. (∘) and 37° C. (Δ).

FIG. 26 shows rheology data (G′: ●, G″:∘) of 2 wt % Max1 solution incell growth media at 37° C. FIG. 26A shows rheology data of gelformation (1% strain, 6 rad/s frequency). FIG. 26B shows frequency sweepdata (5% strain) and FIG. 26C shows G′ and G″ recovery of hydrogel (1%strain, 6 rad/s frequency) after cessation of high amplitude of strain(1000%, 6 rad/s). As shown in FIG. 6 relative to a pH folding andassembly trigger, gels produced by peptide exposure to cell growth mediaform quickly, are significantly rigid, can be shear thinned (e.g.syringe injected), and can quickly reheal to their original stiffnessafter cessation of shear.

FIG. 27 shows a photograph of mesenchymal stem cells encapsulated inhairpin hydrogel network.

FIG. 28 shows a schematic representation of shear thinning and hydrogelreformation.

FIG. 29 shows Encapsulation of Mesenchymal C3H10t1/2 stem cells in MAX1hydrogel. LSCM z-stack image (viewed along the y-axis through the gel,inset) showing the incorporation of cells into a 0.5 wt % MAX1 gelleading to non-homogenous cell encapsulation. Gel-cell constructs areprepared by adding an equal volume suspension of 250K cells in DMEM to a1 wt % buffered peptide solution at pH 7.4, 37° C. Cells are pre-labeledwith cell tracker green to aid visualization. Scale bar is 100 μm.

FIG. 30 shows CD spectroscopy and oscillatory rheology of MAX1 and HPL8hydrogels. FIG. 30 a shows Kinetics of β-sheet formation for MAX1(squares) and HPL8 (triangles). The evolution of β-sheet is monitoredduring the solution-hydrogel phase transition by recording [θ]₂₁₆ as afunction of time for a 0.5 wt % peptide solution at 37° C. after foldingand self-assembly is initiated by the addition of DMEM, pH 7.4. Insetshows C.D. wavelength spectra characteristic of β-sheet structure forMAX1 and HPL8 hydrogels after the kinetics measurements. FIG. 30 b,shows dynamic time sweep measurements of MAX1 (squares) and HPL8(triangles) monitoring the evolution of storage modulus (G′) as afunction of time for 0.5 wt % hydrogel at 37° C. in DMEM, pH 7.4;frequency=6 rad sec⁻¹, strain=0.2%.

FIG. 31 shows TEM micrographs of MAX1 and HPL8 hydrogels. Nanostructureof 0.5 wt % gel network. FIG. 31 a, MAX1 and FIG. 31 b, HPL8 negativelystained with uranyl acetate. Scale bar is 100 nm. The inset showsmagnification of MAX1 and HPL8 fibrils that are ˜3 nm in width. Scalebar is 20 nm.

FIG. 32 shows Encapsulation of Mesenchymal C3H10t1/2 stem cells in HPL8hydrogel. LSCM z-stack image (viewed along the y-axis through the gel,inset) showing the homogenous incorporation of cells into a 0.5 wt %HPL8 gel. Gel-cell constructs are prepared by adding a suspension of250K cells in DMEM to a 1 wt % buffered peptide solution at pH 7.4, 37°C. Cells are pre-labeled with cell tracker green to aid visualization.Scale bar is 100 μm.

FIG. 33 shows Gel recovery kinetics, distribution of cells and cellviability after shear thinning. FIG. 33 a shows gel recovery assessed bymonitoring G′ as a function of time after shear thinning a 0.5 wt % HPL8gel initially prepared from DMEM, pH 7.4. Region (I) shows onset ofgelation as a function of time for the initial gelation event at 0.2%stain, (II) shear thinning of the resulting hydrogel on application of1000% strain, (III) recovery of hydrogel rigidity after reduction ofstrain to 0.2%; frequency=6 rad sec⁻¹ for all measurements. FIG. 33 bshows a LSCM z-stack image (viewed along the y-axis as in FIGS. 29 and32) showing the distribution of cells in a 0.5 wt % HPL8 hydrogel. Thegel-cell construct was initially prepared in a syringe as described inFIG. 5 and shear thinned into a confocal plate for imaging. Inset showsthe syringe loaded with gel-cell construct prior to shear thinning.Cells were prelabed with cell tracker green for visualization. Scale baris 100 μm. FIG. 33 c shows a LSCM z-stack image (viewed along the z-axisthrough the gel, inset) showing a Live/Dead assay of cells after beingshear-thinned delivered at T=3 hr after delivery. Red=dead cells,green=alive cells. Scale bars are 100 μm.

FIG. 34 shows photographs of 0.5 wt % HPL8 hydrogels prepared in asyringe and shear thinned to various surfaces. All images were takenwhile the sample was held vertically showing that the hydrogel stayslocalized at the point of application. HPL8 gels were prepared as statedin the experimental section. a, Borosilicate surface of a confocal plateb, tissue culture treated polystyrene plate c, skin and d, hydrogelapplied to a hood sash showing application of the gel directly to avertical surface.

FIG. 35 shows an LSCM z-stack image (viewed along the z-axis) showing aLive/Dead assay of an 0.5 wt % HPL8 gel/cell construct prepared directlyin the confocal well at T=3 hr. Red=dead cells, green=alive cells. HPL8gel/cell constructs were prepared as stated in the experimental section.Inset of an eye viewing down the z-axis enabling visualization of howthe confocal image is displayed. Scale bar is 100 μm.

FIG. 36 shows oscillatory rheology of 0.5 wt % MAX1 (squares) and HPL8(triangles) hydrogels. MAX1 and HPL8 samples were prepared as stated inthe experimental section a, Dynamic time sweep (DTS) showing the onsetof gelation at 37° C. monitoring the storage modulus, G′ (solid symbols)and loss modulus, G″ (open symbols) as a function of time, frequency=6rad. sec⁻¹, strain=0.2%, gap=0.5 mm. b, Dynamic strain sweep performedimmediately after the DTS at 37° C. monitoring the G′ and G″ as afunction of frequency, strain=0.2%, gap=0.5 mm.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides a self-assemblyhydrogelation strategy that employs small peptides as the gel scaffoldbuilding block. Peptides may be designed to undergo self-assembly onlyafter intramolecular folding into a β-hairpin conformation that iscapable of intermolecular association. The folding event can betriggered by environmental stimuli affording responsive hydrogelationsystems. Using peptides is advantageous due to their quick chemicalsynthesis and the possibility of incorporating novel residues andfunctional epitopes for tailored material and biological properties. Theself-assembled nature of the hydrogel eliminates the need for exogenouscrosslinking agents. The peptide-based hydrogels presented herein arerigid yet easily processed. This hydrogelation strategy will provide acytocompatible gelation system that may be used for potential in vitroand in vivo tissue engineering construct formation. The presentinvention establishes the relationship between peptide structure andultimate hydrogel morphological, rheological, and cell-level biologicalproperties. This has been accomplished by: 1) Gaining a fundamentalunderstanding of the folding and self-assembly process leading tohydrogel formation and how molecular design affects material properties.By rigorously understanding the relationship between designed peptidestructure, the folding/self-assembly process, and the ultimate materialproperties one can produce hydrogels having tailored tissue engineeringproperties. 2) Enhancing the processibility of hairpin-based hydrogelsby designing active intramolecular folding triggers that allow peptidesolutions to undergo hydrogelation on cue. By designing peptides thatfold in response to biologically compatible cues; for example, pH, saltconcentration, cell culturing media, light, body temperature, orspecific ion concentration (e.g., Ca²⁺); one of ordinary skill in theart can design hydrogelation processes for both in vitro and in vivotissue engineering applications. 3) Determining how peptide structureand material properties affect the adhesion and proliferation of modelcell lines (e.g., fibroblast and osteoblast cell lines). The peptidicand porous nature of the β-hairpin hydrogels make these materialscandidates as cytocompatible (non-cytotoxic, promotes cell adhesion andproliferation), easily functionalized substrates for potential bone andsoft tissue engineering applications. Correlations between peptidestructure, hydrogel rigidity and cytocompatibility have beenestablished.

The present invention incorporates one or more of the aforementionedmaterial properties within one fundamental molecular design. Thisalternate strategy employs four fundamental design facets to producehydrogels: 1) Small de novo designed peptides may be used to preparehydrogels. 2) Hydrogels may be constructed via a pure self-assemblymechanism that eliminates the need for exogenous crosslinking agents. 3)The peptides may be designed such that they do not undergo self-assemblyleading to hydrogel scaffold formation unless they are correctlyintramolecularly folded into a targeted conformation. 4) Thisintramolecular folding event, and thus hydrogelation, can be triggeredby specific, environmental stimuli.

FIG. 1 displays one embodiment of the premise of this simple materialconstruction design. Small peptides are designed to be unstructured inaqueous solution until exact solution conditions dictate intramolecularfolding into a β-hairpin conformation. This facially amphiphilic hairpinis amenable to self-assembly leading to a rigid, porous, β-sheet richhydrogel. We propose a nanostructure for the self-assembled state (atright in FIG. 1) consistent with short segments of fibril interspersedwith hydrophobically associated interfibril junctions (See FIG. 7B). Itshould be noted that this morphology is very different than thatobserved from self-associating peptides that afford classicalfibril-based assemblies. In classical systems, peptides associate intolaminated, β-sheet-rich fibrils. The self-assembly process is normallyvery slow (hours to months) and irreversible affording fibrils that canhave micron length dimensions. Examples exist of hydrogels formed uponentanglement of well-developed, ripened fibrils. In contrast, ourproposed peptide system provides a new mechanism in which triggeredintramolecular folding must occur prior to desired intermolecularself-assembly. Thus, hydrogelation can be initiated by a diverse arrayof environmental triggers. The hydrogelation event is very rapid(occurring in seconds depending on conditions) and, if desired, can bedesigned to be completely reversible. Resultant gels are characterizedby nano- to microporous morphology and significant material rigiditydespite being composed of >98% water.

The feasibility of using small peptides to form complex materials iswell demonstrated in the literature. Peptides have been observed toself-assemble into helical ribbons, nanotubes and vesicles,surface-assembled structures and others. The preparation of materialsfrom small peptides is advantageous because they can be chemicallysynthesized rapidly and novel amino acid residues can be incorporated.In addition, the use of orthogonal protection strategies allows forregioselective ligation of chemical moieties to amino acid side chainsaffording conjugates with tailored functions (e.g. cell adhesion). Interms of regioselectivity, the ability to completely and preciselyfunctionalize the monomeric building block of a self-assembling systemis highly desirable when compared to the relatively nonselective methodsused to chemically modify an existing polymer. In one aspect of theinvention, peptides can be designed to fold in response to theirenvironment; taking advantage of this property can lead to smartmaterials that form (or dissolve) on cue. Example of peptides that maybe used in the practice of one or more aspect of the invention include,but are not limited to the following: MAX1 VKVKVKVK V^(D)PPT KVKVKVKVMAX2 VKVKVKVK V^(D)PPT KVKTKVKV MAX3 VKVKVKTK V^(D)PPT KVKTKVKV MAX4KVKVKVKV K^(D)PPS VKVKVKVK MAX5 VKVKVKVK V^(D)PPT KVKEKVKV MAX6 VKVKVKVKV^(D)PPT KVKCKVKV MAX7 VKVKVKVK V^(D)PGT KVKVKVKV MAX8 VKVKVKVK VP^(D)PTKVKVKVKV MAX9 VKVKVKVK VPPT KVKVKVKV (SEQ ID NO:1) MAX10 VKVKVKVKV^(D)P^(D)PT KVKVKVKV MAX11 VKVKKCK V^(D)PPT KVKCKVKV MAX12 VKVKCKVKV^(D)PPT KVCVKVKV MAX13 ISINYRTE I^(D)PPT SINYRTEI MAX14 VKVKVCVKV^(D)PPT CVKVKVKV MAX15 VKVKVCVK V^(D)PPT KVKVCVKV MAX16 VKVKVKVCV^(D)PPT KVKVCVKV MAX17 RGDVKVKVKVK V^(D)PPT KVKVKVKVRGD MAX18 VKVEVKVEV^(D)PPT KVEVKVEV MAX19 VKVKVKVKVK V^(D)PPT KVKVKVKVKV MAX20 VKVKVKVKYNGT KVKVKVKV (SEQ ID NO:2) MAX21 VKVKVK V^(D)PPT KVKVKV MAX22 VKVKVKVKGGGG KVKVKVKV (SEQ ID NO:3) MAX23 VEVEVEVE V^(D)PPT EVEVEVEV MAX24VXVXVXVX V^(D)PPT XVXVXVXV X = Ornithine MAX25 VXVXVXVX V^(D)PPTXVXVXVXV X = Diaminobutyric acid MAX26 VXVXVXVX V^(D)PPT XVXVXVXV X= Diaminopropionic acid MAX27 VYXYXYX Y^(D)PPT XYXYXYXY X = Valine MAX28VRVRVRVR V^(D)PPT RVRVRVRV MAX29 VKVKVKVKVRGDKVKVKVKV (SEQ ID NO:4)MAX30 XKXKXKXK V^(D)PPT KXKXKXKX X = Aminoisobutyric acid MAX31 XKXKXKXKV^(D)PPT KXKXKXKX X = Norvaline MAX32 XKXKXKXK V^(D)PPT KXKXKXKX X= Norleucine MAX33 FKFKFKFK V^(D)PPT KFKFKFKF MAX34 IKIKIKIK V^(D)PPTKIKIKIKI MAX35 HWSFTIKITV^(D)PPTHWSFTIKITIn addition to the amino acids specifically recited above, at anyposition of any of the above peptides indicated with X, each X canindependently be any natural or non-natural amino acid (L or Dstereochemistry) or any analog of an amino acid known to those skilledin the art. In this application, D stereochemistry will be indicated bya superscript before the D amino acid, thus ^(D)P is D-proline.

In some embodiments of the invention, peptides may fit the generalformula VKVKVKVK(XXXX)_(a)KVKVKV(XXXX)_(b)KVKVKVKV (SEQ ID NO:5).Specific examples of this embodiment of the invention include, but arenot limited to, MAX36 (XXXX)_(a) = V^(D)PPT (XXXX)_(b) = K^(D)PPK MAX37(XXXX)_(a) = V^(D)PGT (XXXX)_(b) = K^(D)PGK MAX38 (XXXX)_(a) = V^(D)PGT(XXXX)_(b) = K^(D)PPK MAX39 (XXXX)_(a) = V^(D)PAT (XXXX)_(b) = K^(D)PAKMAX40 (XXXX)_(a) = V^(D)PPT (XXXX)_(b) = K^(D)PGK MAX41 (XXXX)_(a) =V^(D)PPT (XXXX)_(b) KNGK (SEQ ID NO:6) MAX42 (XXXX)_(a) VNGT (XXXX)_(b)= K^(D)PPK (SEQ ID NO:7) MAX43 (XXXX)_(a) = VNGT (XXXX)_(b) = KNGK MAX44(XXXX)_(a) = V^(D)PAT (XXXX)_(b) = K^(D)P^(D)AKIn addition to the amino acids specifically recited above, at anyposition of any of the above peptides indicated with X, each X canindependently be any natural or non-natural amino acid (L or Dstereochemistry) or any analog of an amino acid known to those skilledin the art. Preferably, each (XXXX)_(a) and (XXX)_(b) may comprise asequence capable of forming a turn (e.g., a β-turn).

In some embodiments of the invention, peptides may fit the followinggeneral formulas: MAXX₁ (VK)_(m)V^(D)PPT(KV)_(n) m = 1-100, n = 100MAXX₂ (VK)_(m)VPPT(KV)_(n) m = 1-100, n = 100 (SEQ ID NO:8) MAXX₃(VK)_(m)V^(D)P^(D)PT(KV)_(n) m = 1-100, n = 100 MAXX₄(VK)_(m)GGGG(KV)_(n) m = 1-100, n = 100 (SEQ ID NO:9) MAXX₅(VK)_(m)VP^(D)PT(KV)_(n) m = 1-100, n = 100 MAXX₆ (VK)_(m)YNGT(KV)_(n) m= 1-100, n = 100 (SEQ ID NO:10) MAXX₇ (VK)_(m)VRGD(KV)_(n) m = 1-100, n= 100 (SEQ ID NO:11)Each m and n may independently be from 1-100 and m may or may not equaln.

Among the advantages of the invention are the ease of hydrogelpreparation and the favorable resultant material properties,particularly processibility and morphology. The use of a self-assemblystrategy eliminates the need for chemical crosslinks. Using chemicals toinduce crosslinks is generally non-selective and many crosslinkingreagents are toxic and not easily purified away from the hydrogelscaffold.

In terms of processibility, hydrogels constructed from physicallycrosslinked, self-assembled networks can be responsive to mechanicalshear. This characteristic provides a free flowing suspension during theapplication of shear and complete reformation of the gel network (selfhealing) after cessation of the shear (see Prudhomme, et al., Langmuir1996, 12, 4651-4659, and Nowak, et al., Nature 2002, 417, 424-428). Thiscombination of shear thinning and self-healing allows material formationin a spatially resolved manner. For example, in some embodiments of theinvention, one of ordinary skill in the art could inject (shear thin) ahydrogel pre-formed ex vivo in the presence of desired biologicalconstituents (growth media, growth factors, living cells, etc . . . )into a host where it self heals providing a scaffold for tissueregeneration. In addition, hydrogels formed via self-assembly are notlimited to ex-vivo preparations as described above. The presentinvention provides peptide systems that undergo self-assembly whenexposed to biologically relevant stimuli such as salt, temperature andpH (see Pochan, et al., Journal of the American Chemical Society 2003,125, 11802-11803 and Schneider, et al., Journal of the American ChemicalSociety 2002, 124, 15030-15037). This could allow for the formation ofhydrogel directly in vivo via the injection of a peptide solution.Hydrogel systems in which both ex and in vivo preparations are possibleare most versatile in terms of processibility and are within the scopeof the present invention.

The self-assembly of β-hairpins described herein may provide rigidnetworks composed of minimal solid material (shear moduli, G′, >1000 Pawith ≦2 wt % peptide) providing for a significantly dilute, porousscaffold with no need for additional processing. These gels are porouson both the nanoscale and microscale. This porous character may aid cellmigration within the hydrogel scaffold and allow the diffusion ofnutrients. In one embodiment, the present invention provides correctlydesigned peptides that can undergo rapid, triggered hydrogelationaffording rigid, porous materials that are cytocompatible. Self-assemblycan be triggered by physiologically relevant stimuli resulting inhydrogels that shear thin, making processing of this system versatile.

The proposed hydrogelation system relies on the ability of a peptide toadopt a folded conformation that is amenable to self-assembly (i.e., ifthe peptide isn't correctly folded, it doesn't self-assemble intohydrogel). In addition, it is possible to design peptides that only foldin response to an environmental cue. The result is a peptide system thatwill undergo hydrogelation only in the presence of a desired stimulus.This environmental dependence provides for a hydrogel that is inherently“smart” in that the system will not gel until the individual peptidesare folded. Depending on the nature of the stimulus (trigger),hydrogelation via this process can be designed to be totally reversible;simply removing the stimulus unfolds the peptides that comprise thescaffold resulting in hydrogel dissolution. Several examples ofenvironmental triggers include temperature, pH, ionic strength, specificion binding (e.g., Ca²⁺), and electromagnetic radiation (e.g., light).

In some aspects, the present invention provides a method of producing ahydrogel that may comprise (a) triggering intramolecular peptide foldinginto β-hairpin conformation, and (b) self-assembling of hairpins intohydrogels. The peptides may be designed so as to provide a hydrogelhaving the desired material properties (e.g., porosity,rigidity/modulus, biofunctionality, etc, see FIG. 1).

All of the desired aspects of the hydrogels of the invention may becontrolled by peptide design. Peptides for use in the present inventionmay be small peptides (e.g., from about 10 to about 200 residues, fromabout 10 to about 100 residues, from about 10 to about 75 residues, fromabout 10 to about 50 residues, from about 10 to about 40 residues, fromabout 10 to about 30 residues, from about 10 to about 25 residues, fromabout 10 to about 20 residues, from about 15 to about 200 residues, fromabout 15 to about 100 residues, from about 15 to about 75 residues, fromabout 15 to about 50 residues, from about 15 to about 40 residues, fromabout 15 to about 30 residues, from about 15 to about 25 residues, fromabout 15 to about 20 residues, from about 20 to about 200 residues, fromabout 20 to about 100 residues, from about 20 to about 75 residues, fromabout 20 to about 50 residues, from about 20 to about 40 residues, fromabout 20 to about 30 residues, or from about 20 to about 25 residues).Peptides of the invention may incorporate one or more modified aminoacid residues (e.g., D-amino acids, homologs of naturally occurringamino acids, amino acids with modified side chains, etc). Peptides ofthe invention preferably adopt a secondary structure (e.g., β-hairpinsecondary structure) in response to one or more triggers. Triggers maybe one or more changes in environmental conditions. While specificpeptides will be described later in detail, in one aspect peptides ofthe invention may be composed of high β-sheet propensity residuesflanking an intermittent four residue turn sequence. Polar and apolarresidues may be arranged sequentially in the strand regions to affordamphiphilic surfaces in the folded state.

In some embodiments of the invention, the ability of peptides toself-assemble is dependent upon their unimolecular folded state. Forexample, under folding conditions peptides may adopt a desired secondarystructure (e.g., may adopt an amphiphilic β-hairpin structure where oneface of the hairpin is lined with hydrophobic residues and the otherface is lined with hydrophilic residues). In this example,intramolecular folding is dictated by the alleviation of charge densityon the hydrophilic face upon folding, the formation of intramolecularhydrophobic van der Waals interactions, the formation of intramolecularhydrogen bonds between β-strands within the hairpin, and the turnpropensity of the turn sequence, FIG. 2. Intimate knowledge of thefactors that govern intramolecular folding allows one to design activetriggering mechanisms of the folding event. After intramolecularfolding, subsequent self-assembly of monomeric hairpins is facilitatedfacially by hydrophobic association of the hydrophobic faces of foldedhairpins and laterally via H-bond formation and hydrophobic van derWaals contacts between neighboring hairpins. Detailed knowledge of theseparameters allows one to control the self-assembly process and thus theultimate material properties. All of these design parameters areoutlined below and shown schematically in FIG. 2. One of ordinary skillin the art can appreciate that peptides may be designed so as to adoptother desired secondary structures that result in a configuration ofpeptide residues favoring intermolecular association of the peptides andgel formation.

Peptides for use in the hydrogels of the invention can be constructed tohave any desired characteristics by varying one or more of the followingparameters: 1) electrostatics, for example, by varying the charge withinthe peptide intramolecular folding rate can be varied; 2) Van der Waalsinteractions, for example, constructing peptides having varying a)lateral and facial intermolecular hydrophobic interactions and/or b)intramolecular hydrophobic interactions, allows varying the folding andself-assembly of the peptides as well as the material properties of thehydrogel; 3) hydrogen bonding, for example peptides may be constructedwith varying a) intramolecular and/or b) intermolecular hydrogen bondformation to vary the folding, self-assembly and final materialproperties; and 4) turn sequence, for example, the turn region ofpeptides of the invention may be designed to control folding and thustrigger self-assembly.

The present invention permits one of ordinary skill in the art to designpeptides with desired characteristics (i.e., the appropriateelectrostatics, intra- and intermolecular hydrophobic van der Waalsinteractions, and turn sequence) so as to have the desiredintramolecular folding, intermolecular self-assembly and materialproperties. In some embodiments, electrostatics and/or hydrophobic Vander Waals interactions can be used to design peptides having activeintramolecular folding triggers. Triggers may be a change in one or morecharacteristic of a solution comprising the peptides, for example, pH,salt concentration, specific ion concentration, electromagneticradiation and/or temperature.

In some embodiments, peptides designed as described herein may be usedto produce hydrogels that may be used as non-cytotoxic scaffolds thatsupport cell adhesion (for example, mammalian cell adhesion) andproliferation.

In one particular embodiment, MAX1, a 20-residue peptide, was designedto probe the effects of electrostatics on peptide intramolecularfolding, self-assembly and consequent hydrogel material properties. Thesequence is composed of high β-sheet propensity valine and lysineresidues flanking an intermittent tetrapeptide -V^(D)PPT- designed toadopt type II′ turn structure, FIG. 3A. In addition to incorporatinglocal design elements to stabilize hairpin structure, nonlocal effectswere also considered by arranging the polar and apolar residues flankingthe β-turn in an alternating fashion to favor β-hairpin formation in theself-assembled state. In addition, a β-branched residue was placed atthe i-position of the turn (Val-9) to enforce a trans prolyl amide bondgeometry at the i+1 position. This design element ensures that underfolding conditions, intramolecular folding of monomeric hairpins isfavored prior to self-assembly. A cis prolyl bond, which is designedagainst, could result in the presentation of individual β-strands withineach monomer in an extended conformation. Peptides capable of adoptingboth cis and trans conformers could undergo indiscriminantself-association of extended and correctly folded monomers and may beactively designed against.

The ability of MAX1 to undergo hydrogelation is dependent upon itsunimolecular folded state. Under basic aqueous solution conditions (pH9.0, 125 mM Borate, 10 mM NaCl), where some of the lysine side chains ofMAX1 are neutral, this peptide intramolecularly folds into anamphiphilic β-hairpin (folding can also be triggered at pH 7 underphysiological conditions). One face of the hairpin is lined withhydrophobic valine residues while the other face is lined withhydrophilic lysine residues, FIGS. 2 and 3A. After intramolecularfolding, subsequent self-assembly of hairpins is facilitated (a)laterally via H-bond formation and attractive hydrophobic interactionsbetween distinct hairpins as well as (b) facially by hydrophobicassociation of the valine-rich faces of the folded peptide resulting inhydrogelation, FIG. 3B. Since the charged state of the lysine residuesare controlled by pH, the unimolecular folding is reversible. Loweringthe pH below the intrinsic pKa of the lysine side chains results inintra-strand charge repulsion from neighboring lysines and subsequentunfolding of individual hairpins, ultimately dissolving theself-assembled hydrogel structure. This reversible behavior is uniquewhen compared to β-sheet-rich hydrogels prepared from classicalfibril-based designs. In these systems, the self-assembly process isirreversible. This suggests that the amphiphilic hairpins undergo aself-assembly mechanism that is different from peptides thatirreversibly lead to fibrils. In fact, thorough characterization of ourgelation process from the molecular level through the macroscopicmaterial properties is presented below and indicates that by linking theintramolecular folding of MAX1 to its ability to self-assemble, aresponsive material can be prepared.

Time-dependent circular dichroism (CD) studies support a hydrogelationmechanism consistent with intramolecular hairpin formation followed byself-assembly. FIG. 4A shows that at micromolar concentrations, when thepH is increased from 5.5 (unfolding conditions) to 9.0 (foldingconditions) unstirred solutions of MAX1 undergo a random coil to β-sheettransition taking hours at 20° C. At temperatures >25° C. the foldingand self-assembly of MAX1 occurs on the order of seconds. The CDspectrum at 406 minutes displays a clear minimum at 216 nm indicatingthat MAX1 adopts structure rich in β-sheet. FIG. 4B demonstrates thatthe observed 0216 is concentration-dependent (comparing 0216 at any onetime point for both concentrations), indicating that MAX1 isself-associating and that the rate of assembly increases as theconcentration increases. This behavior is consistent across all spectro-and microscopic methods used to interrogate MAX1. For example, atmicromolar concentrations used for the CD studies shown in FIGS. 4A and4B, self-assembly takes hours at 20° C. At millimolar concentrations,such as those used in the rheological studies described below,self-assembly leading to hydrogel takes minutes at 20° C. Interestingly,the rate of self-assembly is not only concentration dependent but alsodepends on the rate of mixing; vigorously stirred samples adopt sheetstructure within seconds at 20° C.

The reversibility of the folding and self-assembly process wasinvestigated by measuring θ₂₁₆ as a function of pH and time as shown inFIG. 4C. CD of a stirred solution of MAX1 at pH 5.5 indicates randomcoil conformation. Adjusting the pH to 9.0 by the addition of NaOHresults in β-sheet formation as expected. However, subsequent adjustmentof the pH to 6.0 results in complete loss of sheet signal and fullrecovery of random coil signal. This experiment demonstrates that theassembly process is reversible presumably as a consequence ofdeprotonating and re-protonating the lysine side chains resulting inunimolecular folding and unfolding, respectively. Again, this reversiblebehavior is not observed in hydrogels formed from amyloid/prion-likefibrils.

The existence of β-sheet structure within the hydrogel matrix is alsosupported by FTIR spectroscopy, FIG. 5. 1 wt. % solutions of MAX1 in D₂Oat pH 5.5 (where the peptide is soluble) show an Amide I band at 1644cm⁻¹ suggesting that the peptide is unfolded. However, when the pH ofthis solution is adjusted to 9.0 by the addition of NaOD, gelationoccurs and the Amide I band shifts to 1615 cm⁻¹, strongly suggestingthat MAX1 adopts a structure rich in β-sheet.

Bulk rheology experiments exhibit the manifestation of unimolecularfolding and subsequent self-assembly into a gel network by the onset andgrowth of elastic properties. FIG. 6A shows the results of a time sweepexperiment at constant strain and frequency during which the growth ofthe storage modulus (sample rigidity) was monitored after hydrogelationwas initiated for a 2 wt % solution of MAX1. Similar to the rate ofβ-sheet formation observed by CD (FIG. 4B), rheology shows that thefolding and assembly of MAX1 has significantly progressed after 30minutes at 20° C. (gel storage modulus of 600 Pa). Gel formationcontinued to mature after two hours with a doubling of the gel modulusto 1200 Pa. An equilibrium storage modulus of ˜1600 Pa was reached afterseveral hours of gel formation. This equilibrium behavior is clearlyshown by the linear, frequency independent moduli measurements in FIG.6B. For comparison, the storage modulus of strawberry gelatin is ˜50 Pa(data not shown). Rheology indicates that the crossover concentrationseparating a predominantly liquid-like (G″>G′) vs. a predominantlygel-like (G′>G″) response is about 1 wt %. Several examples of hydrogelmoduli from the literature are also shown in FIG. 6B for comparison.

Two hallmarks of a self-assembled gel are to exhibit shear thinning andsubsequent recovery of elastic properties after shearing has ended. Thisdrop in viscosity (shear thinning) results from the disruption ofphysical crosslinks by the application of strain. FIG. 6C clearly showsthat MAX1-derived gels shear-thin, the viscosity drops with increasingstrain-rate. Importantly, self-assembled gels of MAX1 are capable ofquickly reforming after cessation of shear due to the quick relaxationtime of the molecular self-assembly process. Herein lies one majoradvantage of the β-hairpin based hydrogels for potential use in tissueengineering-ease of processibility. Hydrogels can be preformed ex vivounder highly controlled conditions and introduced into a host viasyringe injection/shear thinning.

In FIG. 6D, a time sweep experiment identical to that performed duringthe original gel formation was run immediately after the application of1000% strain at 6 Hz for 180 seconds. The initial modulus of thereforming gel was 650 Pa with 80% of the equilibrium modulus recoveredafter only 30 minutes making this a quick recovering and relativelystrong hydrogel. For comparison, a 2 wt % gelatin hydrogel takes over 4h to recover 80% of its equilibrium modulus after shear thinning.

An interesting comparison can be made between the rheology data and theconcentration dependent CD data in FIG. 4B. CD clearly demonstrates thatthe rate at which MAX1 intramolecularly folds and self-assembles ispositively dependent on peptide concentration. The rheologicallymonitored formation of the 2.0 wt % gel, approximately 6 mM inconcentration, can be considered an analogous view of molecular foldingand self-assembly at higher peptide concentrations, FIG. 6A. Only afterfolding can the β-hairpins self-assemble into a gel scaffold and thusprovide a viscoelastic response. In fact, a control peptide similar insequence to MAX1 but designed to disfavor hairpin formation did notundergo hydrogelation when subjected to identical folding conditions(see below).

A rheology experiment demonstrating that the gelation mechanism isreversible with pH changes was attempted by adding a small amount ofconcentrated HCl to a 2 wt % gel resulting in a final pH ˜6.0. Therheological response of the system was essentially that of pure water,below the sensitivity threshold of the instrument. This indicated aclear obviation of the self-assembled scaffold and reversibility ofgelation. This is in complete agreement with the immediate unfolding ofMAX1 under acidic conditions as actively monitored via CD, FIG. 4C.Therefore, CD experiments, which actively monitored the intramolecularfolding and intermolecular sheet assembly of MAX1, and rheologyexperiments, which actively monitored the self-assembly of peptide intoa gel scaffold, combine to form a clear image of how material propertiescan be attributed to molecular folding and consequent assemblymechanisms.

Laser scanning confocal microscopy (LSCM) reveals a heterogeneous gelmicrostructure in which a continuous gel matrix is permeated by waterchannels, FIG. 7A. This microscale porosity may be exploited to producea tissue engineering scaffold. Porous microstructure must be processedinto hydrogels prepared from traditional crosslinked hydrophilicpolymers. In contrast, the microporosity of hairpin-based gels is aresult of the self-assembly process and additional processing is notnecessary. The gel matrix regions in FIG. 7A are not solid peptide butrather a dilute peptide network on the nanoscale that is itselfpermeated by water, FIG. 7B.

Combined small and ultrasmall neutron scattering (SANS/USANS) data for a1 wt % gel in D₂O (FIG. 8) is consistent with fibrillar structure of thegel matrix on the nanoscale and the heterogeneous morphology on themicroscale. Intensity at low scattering vector, q (whereq=(4π/λ)sin(θ/2), λ=neutron wavelength and θ=scattering angle), clearlyexhibits a slope of −4 indicative of scattering from a sharp interfacebetween two phases. In this q regime the two phases are the gel matrixand the 1-10 μm sized water pores and channels as clearly observed inthe LSCM in FIG. 7A. In the SANS data at higher q, the most notablefeature is the slope of approximately −1 in the Gunier region where theproduct of q and the radius of gyration, R_(g), of a particle is <1.This is indicative of scattering from a rod-like object on thenanoscale. This description is consistent with what can be observed inthe cryoTEM data (FIG. 7B) where the gel scaffold consists of shortregions of fibrillar structure interconnected via junction points.

The results discussed above allow one of ordinary skill in the art tovary the electrostatic characteristics of a peptide and thereby controlthe formation and/or material properties of hydrogels of the invention.In the specific example described above, self-assembly and materialproperties demonstrate that peptide folding is partly governed byreducing the lysine charge density of the primary sequence. This allowsall of the lysine residues to occupy one face of the folded amphiphilichairpin. One of ordinary skill in the art will appreciate that aminoacid residues other than lysine may be used. For example, any residuethat has a charge or can be made to have a charge by varying theenvironmental conditions may be used. In addition, multiple differentamino acid residues may be used in the same peptide.

In some embodiments of the invention, by using changes in solution pH,the intramolecular folding event and subsequent hydrogelation can betriggered and may be reversible. Thus, chemical responsiveness may berealized by linking intramolecular folding to consequent intermolecularassembly. Resultant hydrogels may be mechanically rigid, yet porous onthe nano- and microscale, making them very good candidates ascytocompatible materials. In addition, the self-assembled nature of thehydrogel scaffold produces mechanically responsive materials, anattribute that could be exploited to deliver hydrogel/cell constructsvia shear thinning methodology.

With reference to FIG. 2A, the present invention demonstrates that theformation of intramolecular Van der Waals contacts between residueswithin a hairpin may stabilize the folded state and that intermolecularlateral and facial contacts between residue side chains of distincthairpins may stabilize the self-assembled state. This allows thecharacteristics of the hydrogels of the invention to be varied byadjusting these parameters. This is demonstrated by studying thetemperature dependence of hairpin folding and self-assembly of peptidesthat vary in relative hydrophobicity. It is well known that water isbetter able to solvate hydrophobic groups at low temperatures. Thisphenomenon has been used to describe the cold denaturation of proteinswhere some proteins unfold at low temperatures, exposing theirhydrophobic interiors to aqueous solvent. A 150 μM aqueous solution ofMAX1 unfolds when cooled below room temperature. The CD spectrum at 5°C. is consistent with random coil conformation, FIG. 9A. However,warming the solution to 40° C. results in a spectra consistent withβ-sheet structure. Monitoring the mean residue ellipticity at 218 nm asa function of temperature demonstrates that the temperature at whichfolding and consequent self-assembly is triggered (T_(gel)) isapproximately 20° C., FIG. 9B. In fact, previous studies involving thepH induced folding and self-assembly of MAX1 were performed at 20° C. Atthis temperature, folding and self-assembly are slow enough to beconveniently monitored. At higher temperatures, folding andself-assembly leading to hydrogelation is fast (instantaneous to secondsdepending on concentration). If the formation of hydrophobicinteractions affects the folding and self-assembly process, thenaltering the hydrophobic content of MAX1 should affect the temperatureat which folding is triggered. FIG. 9B shows CD data for MAX1 and twoadditional peptides, MAX2 and MAX3. MAX2 is identical to MAX1 exceptthat one valine residue has been replaced with the isostructural, butless hydrophobic residue, threonine. In MAX3, two valines are replacedwith threonine residues. The resulting peptides vary in theirhydrophobicity as evident by comparing calculated free energies oftransfer from octanol into water, FIG. 9. CD data shows that as thehydrophobicity decreases, the temperature at which folding is inducedincrementally increases. Thus, the formation of hydrophobic contactsinfluences folding and self-assembly. This data also shows that inaddition to pH, temperature can be used to trigger folding. Thus, thecharacteristics of the hydrogels of the invention can be varied byadjusting the amino acid residues of the peptides used to form thehydrogels to increase or decrease the hydrophobicity of the amino acidresidues used.

There are reports of large polymers that are thermally responsive. Thepresent invention provides the first system that employs temperaturetriggered folding to induce self-assembly. Although thermally inducedhydrogelation is not reversible for MAX1 and MAX2, the folding,self-assembly and consequent hydrogelation of MAX3 is thermallyreversible. CD spectra of a 150 μM solution of MAX3 demonstrates thatthis peptide is unfolded at 5° C., FIG. 10A. Heating the solution to 80°C. results in a spectra consistent with β-sheet structure. Subsequenttemperature cycles show that folding and unfolding are reversible. InFIG. 10B, rheology demonstrates that a 2 wt % aqueous solution of MAX3undergoes thermally reversible hydrogelation over severalheating/cooling cycles. The temperatures used in both CD and rheologybracket the temperature (T_(gel)=60° C., FIG. 9) at which folding andconsequent self-assembly is triggered. The CD and rheology data takentogether suggest a mechanism of hydrogelation consistent withtemperature induced unimolecular folding followed by self-assembly. Thetemperature responsive behavior exhibited by these peptides expandstheir versatility due to ease of processing. For example, free flowingpeptide solutions could be prepared at room temperature and administeredin vivo where the temperature of the body could induce gelation. SomePoly-N-isopropylacrylamide polymers have been engineered have beenengineered to undergo such transitions ultimately affordingextracellular-like scaffolds for tissue regeneration. The above studiessupport the idea that the formation of hydrophobic interactions areimportant. MAX1-3 all contain valines and threonines at hydrogen bondedpositions. At these positions, their side chains prefer to adopt a transrotamer. As a result, hydrophobic residues that oppose each other acrossthe hairpin point their side chains in opposite directions thus makingthe formation of intramolecular hydrophobic interactions across thestrands difficult, FIG. 11A. However, these side chains are nicelypositioned to interact laterally with the hydrophobic side chains ofneighboring hairpins in the self-assembled state, FIG. 11B. Formingthese lateral hydrophobic interactions should favor self-assembly. Thiswas demonstrated using MAX4, a peptide of comparable hydrophobicity toMAX1 that incorporates all of its valine residues at nonhydrogen bondedpositions. In MAX4, opposing valine side chains are expected to pointtowards each other, FIG. 11B. Thus, the valine side chains of MAX4 areless likely than those in MAX1 to form lateral intermolecularhydrophobic interactions. If the formation of lateral intermolecularhydrophobic interactions is of more importance for self-assembly thanintramolecular hydrophobic contacts, then the rate of self-assemblyshould be faster for MAX1 than for MAX4. This difference in rate shouldbe evident in the rate of storage moduli (rigidity) increase. Indeed,FIG. 12 shows that self-assembly leading to a rheological response isfaster for 1 wt % preparations of MAX1 as compared to MAX4 at 40° C.(T_(gel)=20° C. for both peptides). Therefore, in addition toelectrostatics, the formation of lateral hydrophobic contacts duringself-assembly also contributes to hydrogelation.

In some embodiments of the invention, various functionalities (e.g.,cell adhesion epitopes, receptor agonists, receptor antagonists,ligands, small molecules, etc) may be incorporated into the hairpinhydrogel scaffold. One way to accomplish this would be to functionalizeone or more amino acid side chains of the peptide. MAX2 and MAX3, whichcontain threonine substitutions on the hydrophobic face, are capable offolding and self-assembling at elevated temperatures indicating thatneutral residues having similarly sized side chains may be tolerated. Insome embodiments, the moieties to be incorporated at a particular sidechain may be much larger (e.g. RGD-based motifs) and may contain chargedresidues. MAX5 (VKVKVKVKV^(D)PPTKVKEKVKV-NH₂) was prepared toinvestigate the possibility of incorporating a charged residue on thehydrophobic face of MAX1. The sequence contains a glutamic acid residueat position 16 which is negatively charged at basic pH where MAX1normally folds and assembles. CD spectroscopy confirmed that 150 μMsolutions (pH 9.0, 125 mM Borate, 10 mM NaCl) of MAX5 exist as randomcoil indicating that negative charges are not tolerated on thehydrophobic face (data not shown). However, alterations to thehydrophilic face of MAX1 are well tolerated. Sequences in which one ortwo lysine residues are replaced with other residues such as cysteine,serine, and glutamic acid are capable of folding and self-assembly intorigid hydrogels (data not shown).

The peptides described above contain identical turn sequences, namely(-V^(D)PPT-). This sequence has a strong propensity to form a type II′turn and therefore helps to drive intramolecular folding. Sinceintramolecular folding must occur prior to self-assembly, alterations tothis turn sequence that inhibit turn structure formation can be used tomodulate intramolecular folding and self-assembly leading tohydrogelation. MAX9 was prepared to probe the importance of the turnregion (VKVKVKVKV^(L)PPTKVKVKVKV-NH₂). MAX9 is identical to MAX1 withthe exception that the ^(D)Pro at position ten has been replaced with^(L)Pro. Unlike the dipeptide ^(D)Pro^(L)Pro contained within MAX1 thatfavors type II′ turn formation, the ^(L)Pro^(L)Pro motif of MAX9 favorsan open conformation. The two strands emanating from an open^(L)Pro^(L)Pro conformation would be projected in opposite directions.Thus, intramolecular folding resulting in β-hairpin formation would behighly unfavored. Any observable self-assembly would likely result fromthe direct intermolecular association of extended peptide conformers. CDof a 150 μM solution of MAX9 under folding conditions showed only randomcoil even after four hours (data not shown). Also, 2 wt % solutions ofMAX9 failed to undergo hydrogelation and remained a low viscositysolution for days. Interestingly, after one week, self-assembly didoccur but did not result in hydrogelation. Instead, long fibrils wereobserved that have dimensions consistent with self-assembled, extendedconformers of MAX9, FIG. 13. This is strong support that intramolecularfolding into a β-hairpin conformation is necessary for self-assemblyinto hydrogel and that the mechanism leading to hydrogel is distinctfrom the mechanism leading to extended fibril. In some embodiments, turnsequences can be incorporated that serve as cell adhesion sites. Forexample RGD binding epitopes are commonly found within turn regions ofproteins important in cell adhesion events.

In one aspect, the present invention provides peptides having designedsequences that fold only in the presence of a desired environmentalstimulus resulting in a hydrogelation system that is triggered on cue.The ability to control material formation temporally and spatiallyallows one to fully control the processing of the material. Specific topreparing hydrogel-cell constructs for tissue engineering applications,controllable hydrogel formation can take place in vitro and/or in vivodepending on the nature of the triggering mechanism. For example, MAX1is unfolded under acidic solution conditions (pH<9, 10 mM NaCl) butrapidly folds and self-assembles within seconds when the pH is adjustedto 9 (10 mM NaCl) at temperatures greater than 25° C. Peptides were alsopresented that fold in response to changes in temperature. For exampleMAX2 is unfolded at pH 9 (10 mM NaCl) when the temperature is below 40°C., but folds/self-assembles when warmed to temperatures greater than45° C., FIG. 9B. We have also demonstrated that the temperature at whichfolding occurs can be predictably modulated. It is important to pointout that designing folding triggers is only made possible byunderstanding the fundamental principles that govern the folding andself-assembly process.

Hydrogelation of peptides discussed thus far has occurred at basicsolution conditions (pH=9) in the presence of 10 mM NaCl. It isdesirable to trigger hydrogelation at biologically relevant conditions,namely pH 7 in the presence of 150 mM NaCl. In fact, initiating foldingand self-assembly at these solution conditions is possible with MAX1.FIG. 14A shows CD data indicating that at pH 7 (20 mM Tris) the foldingof MAX1 is dependent on salt. In the presence of 20 mM KF, MAX1 isunfolded, but in the presence of 150 mM KF, MAX1 folds andself-assembles. Optically transparent KF is used for CD studies since150 mM solutions of NaCl produce significant signal scatter at lowwavelengths. The salt triggered folding event is further confirmed byFTIR which shows that the amide I band at 1643 cm⁻¹ (random coil) shiftsto 1616 cm⁻¹ (1-sheet) upon the addition of KF at pH 7 for 1 wt %preparations (data not shown). NaCl is also capable of triggeringhydrogelation at pH 7, adding NaCl (final concentration=150 mM) to anaqueous solution of MAX1 results in self-assembly affording rigidhydrogels (3000 Pa at 2 wt %, data not shown). For the formation ofhydrogel-cell constructs, initiating hydrogelation by the addition ofcell growth media would be ideal. Since DMEM growth media contains NaClas well as other salts (˜400 mM in total salt concentration) it can beused as a trigger. 2 wt. % aqueous solutions of MAX1 will undergohydrogelation on the addition of serum free DMEM growth media. FIG. 14Bshows frequency sweep data for the resultant viscoelastic, rigid gels.Additional folding triggers may be used to initiate folding including,but not limited to, specific ion binding (e.g., calcium binding) and/orelectromagnetic radiation (e.g., light).

Those skilled in the art are aware that the mechanical properties ofmaterial substrates affect cell-material interactions. For example, ithas been recently shown that vascular smooth muscle cells migrate fromless rigid to more rigid areas of crosslinked polyacrylamide substrata.Also, a direct correlation between the rate of dorsal root ganglionneurite extension and agarose hydrogel rigidity has been shown. Thepresent invention allows the modulation of the rigidity of hydrogels(e.g., MAX1 containing hydrogels) via peptide concentration and peptidefolding triggering conditions, namely salt concentration andtemperature. FIG. 15 shows the rigidity of 2 wt % MAX1 hydrogels atconstant salt concentration and pH 7.4 assembled at differenttemperatures. The bulk modulus of the final networks is tunable over 3decades from low to high temperature. Importantly, when MAX1 assembly istriggered with temperature the process is irreversible and thus the gelstiffness achieved at each triggering temperature is maintained whentaken to physiological temperature. The tunability of hydrogel stiffnesswill allow correlation between hydrogel rigidity and cellular behaviorsuch as adhesion and proliferation.

The present invention demonstrates that the folding triggers describedherein can be used to form hydrogels that support the adhesion andproliferation of fibroblasts (NIH 3T3, mouse). These cells were used asa model system because of their importance in connective tissuedevelopment and their easily distinguished morphology when adhered tothe hydrogel scaffold. Qualitative studies were performed in which cellswere added to either polystyrene control wells or wells containing auniform slab (3 mm thick) of MAX1-hydrogel. Cell adhesion wasqualitatively monitored via direct observation of cell spreading afterfour hours from the time of initial cell seeding by optical microscopy(a quantitative cell attachment assay is described below, see alsoAkiyama, S. K. Functional analysis of cell adhesion: Quantitation ofcell-matrix attachment, 2002; Vol. 69, pp 281-296). Non-adheredfibroblasts have rounded morphologies and adhered fibroblasts havespread morphologies Proliferation was measured both qualitatively byoptical microscopy and quantitatively by a ³H thymidine-based cellseeding assay as described below. Hydrogels were prepared in 48 welltissue culture plates by either one of the following protocols. Protocol1: MAX1 was dissolved in water (resulting in an acidic solution, pH˜5)and an equal volume of buffer (pH 9, 250 mM borate, 20 mM NaCl) wasadded at room temperature to induce folding/self-assembly resulting in a2 wt % hydrogel. The resulting gel was bathed in DMEM containing 10%calf serum and 10 mg/mL gentamicin. This allowed media to permeatethroughout the gel and adjust the pH to 7.3 The storage moduli(rigidity) of gels prepared by protocol 1 are about 1600 Pa. Protocol 2:MAX1 was dissolved in water and an equal volume of serum free DMEMcontaining 10 mg/mL gentamicin was added to induce folding/self-assemblyaffording 2 wt % hydrogels. Depending on the experiment, DMEM containingcalf serum can then be added to introduce serum proteins. The storagemoduli of gels prepared by protocol 2 are about 1200 Pa. FIG. 16A showsthat after four hours (in the presence of serum) fibroblasts haveadhered to the surface of the gel as indicated by their spread outmorphology. Proliferation of the cells occurs until confluency isreached, usually around 72 hours if 10⁴ cells are initially plated, FIG.16B. Cells remain viable for at least a month as long as fresh media isprovided (we stopped the assay after 1 month). Cells added to controlwells containing no gel show similar behavior, FIG. 16 C and D. In FIGS.16A-D, cells are plated in the presence of DMEM containing 10% calfserum. Serum proteins may help mediate cellular adhesion to the hydrogelby first coating the scaffold. To determine if the hydrogel scaffoldalone is conducive to cell adhesion, cells were plated onto 2 wt. %hydrogels in the absence of serum. FIG. 16E shows that the fibroblastsbegin to attach and spread after about four hours even in the absence ofserum proteins, but to a lesser extent as compared to the serumcontaining cultures. Addition of serum after four hours, results in cellproliferation, reaching near confluency after 72 hours, FIG. 16F.Identical control experiments performed in wells without hydrogel,showed that the cells behaved similarly. These experiments demonstratethat the peptidic surface provided by the hydrogel scaffold is amenableto fibroblast adhesion in the presence of serum proteins and to a lesserextent when serum is not present. The incorporation of cell bindingepitopes in peptides used in hydrogel formation may be used to enhancecell adhesion in the absence of serum. This optical microscopy dataqualitatively shows that MAX1 hydrogels support fibroblastproliferation.

A quantitative assay that could be used to measure the rate of cellproliferation was developed. This allows gels having different materialproperties and constituent peptide sequences to be compared in terms ofhow well they support proliferation. Standard colorimetric assays suchas the MTT and XTT have proven unsuitable for use with hydrogels of theinvention. These assays rely on the ability of cells to metabolizetetrazolium derivatives to their corresponding colored formazan analogs.The analogs permeate into the hairpin hydrogel and become immobilizedmaking subsequent quantification by UV spectroscopy unreliable. Thisproblem has been addressed by utilizing ³H thymidine-based assays inwhich thymidine is incorporated into proliferating cells undergoing DNAreplication. This method does not require the solubilization of ananalyte and unincorporated thymidine is easily washed from the hydrogelscaffold making quantification of only incorporated thymidine possible.The assay is two-fold. First, a cell seeding experiment may be performedto determine the optimal number of cells that should be plated if onewishes to follow proliferation over a given time period without thecells reaching confluency (48 hours is chosen here for convenience).Second, a proliferation rate may be determined for a given material byseeding the cells (at optimal density) onto the material and determiningthe number of cells undergoing DNA replication at discrete time pointsup to 48 hours when confluency is reached. Shown in FIG. 17 are resultsfrom a ³H thymidine cell seeding experiment that allows one to determinethe optimal cell seeding density for hydrogels of the invention. Cellsto be assayed (e.g., fibroblasts) may be seeded onto either MAX1hydrogels or a polystyrene control surface at different initialdensities. After 48 hours, the growth media can be removed and replacedwith media containing ³H thymidine and incubated for three hours.Unincorporated thymidine may be washed out (washes may be assayed until3H thymidine is no longer detected) and the cells sacrificed forscintillation counting. The data shows that for 2 day proliferationassays involving MAX1 hydrogels, 40,000 fibroblasts should be seededproviding an optimal rate of proliferation. It should be noted that theoptimal seeding density for polystyrene is different (20,000); it isknown that any given cell type will display different optimal seedingdensities for distinct materials (and different cell types will havedifferent optimal seeding densities for one given material). Therefore,for each new material, similar cell seeding experiments may be performedbefore rates of proliferation are determined. This data also suggeststhat after 48 hours, more cells have grown on the hydrogel than on thecontrol surface (comparing degradations (counts) at each surface'srespective optimal seeding density). A possible explanation is that thethree dimensional porosity of the hydrogel scaffold allows cells toproliferate into the hydrogel, which is not possible with a twodimensional polystyrene surface. This data shows that a quantitativeproliferation assay has been established. One of ordinary skill in theart can use the assay described herein to study the effects of peptidesequence and hydrogel rigidity on cell proliferation.

The experiments outlined in FIGS. 16 and 17 entail plating the cellsonto the two-dimensional surface of gel. Assays accessing adhesion andproliferation on the surface of two dimensional gels allow facilecomparison among different hydrogel scaffolds and the results can bedirectly compared to materials in the literature since this is the mostcommon technique to investigate material-cell interactions.

The present invention also encompasses hydrogel-cell constructs in whichcells are incorporated throughout the gel scaffold in three dimensions.This can be accomplished by initiating the hydrogelation of aqueoussolutions of MAX1 via the addition of serum free DMEM that containscells, e.g., stem cells, osteoblasts or fibroblasts. Scaffolds thatmimic a cell's 3-D in vivo environment will provide additional insightsinto cell-material interactions not evident in two dimensional studies.FIG. 18 shows an LSCM image of a 2 wt. % MAX1 hydrogel prepared in thismanner. The image is constructed so that one can view the interior ofthe gel from the side. Cells stained with cell tracker green (CMFDA,Molecular Probes) are seen throughout the gel with a slightly largerconcentration near the bottom of the gel where cells begin to settlebefore gelation is complete. One of ordinary skill in the art willappreciate that any cell type of interest may be used in conjunctionwith the present invention. For example, cells that may be used include,but are not limited to, yeast cells, plant cells and animal cells.Suitable cells are commercially available from, for example, knownculture depositories, e.g., American Type Culture Collection (Manassas,Va.), and commercial sources known to those in the art. Preferred animalcells for use in the methods of the invention include, but are notlimited to, insect cells (most preferably Drosophila cells, Spodopteracells and Trichoplusia cells), nematode cells (most preferably C.elegans cells) and mammalian cells (including but not limited to CHOcells, COS cells, VERO cells, BHK cells, AE-1 cells, SP2/0 cells, L5.1cells, hybridoma cells and most preferably human cells such as 293cells, PER-C6 cells and HeLa cells). In addition, primary cell cultures,tissue homogenates, and/or cells derived from tissue homogenates may beused in conjunction with the present invention.

With regard to FIG. 2, electrostatics, H-bonding and hydrophobicinteractions all play a role in folding and self-assembly. Varyingelectrostatics and lateral inter- and -intramolecular hydrophobiccontacts for folding and self-assembly can be used to vary the materialproperties and/or formation conditions of the hydrogels of theinvention. In addition, varying the turn region of the hairpin may alsobe used to modulate the characteristics of the hydrogels of theinvention. Without wishing to be bound by theory, it is believed thatself-assembly is governed by the interplay of two factors: 1)hydrophobic collapse of hairpins; and 2) intermolecular H-bondformation. The interplay of these two factors results in scaffoldscomposed of short segments of fibril interspersed with hydrophobicallyassociated interfibril junctions, FIG. 1. One of ordinary skill in theart, given the teachings set forth herein, can alter the sequences (andhence physical nature) of the peptides used in order to modulate thecontribution of either or both of these factors in order to produce ahydrogel having any desired characteristics.

The intermolecular interactions outlined in FIG. 2 may be varied tocontrol the self-assembly process and resultant material properties,such as the degree of gel porosity which is important for cellproliferation. Specific examples of hairpins composed of 20 residueshave been provided herein. The present invention also encompassespeptides both longer and shorter than this and the length of thepeptides used may be varied so as to influence material properties ofthe resulting hydrogels.

The present invention also encompasses peptides having one or more aminoacid substitutions as compared to the turns of the peptides used in thespecific examples. Any turn sequence know to those skilled in the artmay be used in conjunction with the present invention.

In some embodiments of the invention, one or more functional moieties,which may be peptide sequences, organic molecules or other molecules,which may be incorporated into the peptides at, for example, the sidechains of the amino acids emanating from the hydrophilic face, the sidechains of the amino acids emanating from the hydrophobic face, the sidechains of the amino acids comprising the turn sequence, within theprimary sequence of one or both strands of the hairpin, and/or withinthe primary sequence of the turn sequence. Specific examples of suchfunctional moieties include, but are not limited to, peptide sequences(e.g., cell adhesion epitopes, nuclear localization signals, etc), andreceptor agonists and/or receptor antagonists (e.g., cholesterolderivatives and the like), peptidomimetics, cyclic peptides, metalchelators, fluorescent and spin active probes and small moleculetherapeutics. Peptides comprising one or more of such functionalmoieties may be analyzed by spectroscopy, light and neutron scattering,microscopy, and rheology techniques as described herein in order toproduce a hydrogel having the folding/self-assembly characteristicsand/or material properties desired.

One or more amino acids of the peptides of the invention may be modifiedduring and/or after peptide synthesis. For example, polyethylene glycol(PEG) derivatives of peptides of the invention may be prepared and, forexample, used for cell culturing and encapsulation. Peptides for use inthe invention may be modified (i.e., derivatized) in any combination ofat their termini, main chain atoms and/or side-chain atoms. More thanone modification may be made and such modifications may be the same ordifferent. In a specific embodiment, PEG units ranging in molecularweight from about 60- to about 4000 may be used to prepare derivativesof peptides for use in the invention. PEG-incorporation may be used tomodulate biocompatibility and hydrogel mechanical properties.

In one embodiment, peptides of the invention may comprise one or morechemical crosslinks and such peptides may be used to prepare hydrogelsof the invention. Chemically crosslinked hydrogels may be used in any ofthe methods of the invention. For example, chemically crosslinkedhydrogels may be used in delivering therapeutic agents, cell culturingand encapsulation. Typically, chemical crosslinks may be formed betweenany side-chain or main chain atom of a peptide with any other side-chainor main chain atom of the same or a different peptide. Crosslinking maybe performed before, during or after self-assembly to fine-tunemechanical and biological properties of resulting hydrogels.

Any technique known to those skilled in the art may be used to introduceone or more crosslinks into one peptide and/or between two or morepeptides in a hydrogel of the invention. In general, anyreactive-group-bearing side chain (e.g., nucleophilic-group-bearingand/or electrophilic-group-bearing side chain) can be crosslinked withsuitable bifunctional molecules (e.g., electrophilic molecules and/ornucleophilic molecules). Suitable crosslinking reagents are commerciallyavailable, from, for example Pierce Biotechnology, Inc., P.O. Box 117,Rockford, Ill. 61105 U.S.A.

In some embodiments, two or more reactive groups may be incorporatedinto a peptide. When the peptide is folded in response to anenvironmental stimulus, the reactive groups may be brought intoproximity and may react with each other forming an intramolecularcrosslink. For example, a peptide may contain two cysteine residues thatare brought together to form a disulfide bond.

Optionally, a crosslinking agent may be added to link the reactive sitesbefore or after addition of the environmental stimulus. For example,peptides may contain one or more reactive groups and reactive groups indifferent peptides may be crosslinked forming an intermolecularcrosslink. In yet another embodiment, a peptide may contain reactivegroups that form intramolecular crosslinks as well as intermolecularcrosslinks. In this case, the reactive groups forming intramolecularcrosslinks may be the same or different as those forming intermolecularcrosslinks. Further, crosslinks may be formed in any order, for example,intramolecular crosslinks may be formed before, after or at the sametime as intermolecular crosslinks.

In some embodiments, reactive groups on different peptides may becrosslinked. For example, peptides may be prepared that comprise one ormore cysteines which may react with cysteines on a different peptide toform an intermolecular disulfide bond. In other embodiments,crosslinking reagents may be utilized to crosslink cysteine-containingpeptides. Suitable crosslinking reagents include, but are not limitedto, BM[PEO]₄ (1,11-bis-Maleimidotetraethyleneglycol) and BM[PEO]₃(1,8-bis-Maleimidotriethyleneglycol).

In some embodiments, lysine-based crosslinks can be installed byreacting the amine side chains of the Lys residues with a suitablecrosslinking agent, e.g., a linear diacid. Suitable crosslinkingreagents may include, but are not limited to, EGS (Ethylene glycolbis[succinimidylsuccinate]) and DTSSP(3,3′-Dithiobis[sulfosuccinimidylpropionate]). In another embodiment,cysteine side chains may be utilized to induce crosslinking.

In some embodiments, crosslinks may be installed by enzymatic reactions.For example, an enzyme capable of linking reactive groups may beutilized to link the side chains of the peptides. In some cases, enzymesmay directly utilize side chains as substrates to create a side chain toside chain crosslink. In other embodiments, an additional substrate maybe included to create a side chain-substrate-side chain crosslink.

In order to vary the contribution of forming intermolecular facial andlateral hydrophobic contacts relative to constant H-bonding, peptidesbased on MAX1 may be prepared. Such peptides may have the same number ofresidues but contain amino acids of varying side chain hydrophobicity onthe hydrophobic face of the folded hairpin. Thus, the number of possibleintra- and intermolecular H-bonds formed during folding andself-assembly is constant but the surface area of the hydrophobic facevaries. Examples of peptides that may be prepared include, but are notlimited to, those of the general sequence XKXKXKXKV^(D)PPTKXKXKXKX-NH₂where X=valine, MAX1; X=isoleucine, MAX7; and X=phenylalanine, MAX8.Molecular modeling (Insight/Discover) indicates that the surface areasof these peptides increase in the order MAX1<MAX7<MAX8. Varying thesurface area may effect various parameters of the hydrogels, forexample, may decrease the temperature at which each peptide folds andself-assembles (T_(gel)) as monitored by CD (see FIG. 9). Also, therheological properties of each peptide may be different; morehydrophobic peptides may lead to more rigid hydrogels. In addition, therate of hydrogelation may be affected with more hydrophobic peptidesforming hydrogels faster. Rate of hydrogelation can be determined byrheology (see FIG. 12). Cryo-TEM can be used to determine if thesedesign changes affect the nano-scale structure of the hydrogel (e.g. asthe hydrophobic surface area increases the number of interfibriljunctions in the self-assembled state may increase) and LSCM can beemployed to assess changes in micro-scale structure (e.g., changes ingel porosity). Varying the nature and number of amino acid residuesemanating from the hydrophobic face of the folded peptides and/orvarying the nature and number of functional moieties emanating from thehydrophobic face of the folded peptides may be used to vary the materialcharacteristics of the hydrogels thus formed.

In order to vary the contribution of forming intermolecular hydrogenbonds during self-assembly, peptides may be prepared that have a greateror lesser capability of forming intermolecular hydrogen bonds. Forexample, derivatives of MAX1 containing Nα-alkylated lysine residues maybe prepared. Lysine residues in MAX1 are sequentially positioned suchthat they are able to form intermolecular H-bonds with neighboringhairpins during self-assembly, FIG. 3A. Alkylating one or more lysineresidues within each strand of a hairpin may inhibit self-assemblyleading to hydrogelation. Examples of such peptides include, but are notlimited to, MAX9 (VKVKVKVKV^(D)PPTKVKVKVKV-NH₂) and MAX10(VKVKVKVKV^(D)PPTKVKVKVKV-NH₂), which contain Nα-butylated lysineresidues at the bold positions. One of ordinary skill in the art willreadily appreciate that by varying the amino acid residues and/orfunctional moieties attached to the amino acid residues, other peptideshaving a modulated (increased or decreased) capability of formingintermolecular hydrogen bonds may be constructed. It is known that theself-assembly of human amylin into fibrils has been inhibited bypeptides containing N-butyl residues. CD spectroscopy and rheology maybe used to assess the ability of these peptides to fold andself-assemble into hydrogel under varying folding conditions.

In some embodiments, peptides may be constructed so as to take advantageof intermolecular interactions to control the self-assembly process andresultant material properties. For peptides that fold and self-assemblevia a combination of hydrophobic collapse and intermolecular H-bonding,temperature may be used to control the degree to which each of theseprocesses contributes to the self-assembly mechanism. As discussedabove, each peptide has a characteristic temperature at which it folds(T_(gel)) and begins to self-assemble. In addition, it has been welldemonstrated in the literature that temperature can be used to drivehydrophobic collapse. Therefore, for any given peptide, if just enoughheat is supplied to fold but not greatly enhance hydrophobic collapse,then H-bonding may predominate the self-assembly mechanism and resultinghydrogel scaffolds may be composed of longer segments of fibrilstructure. Resultant hydrogels may be characterized by larger poresizes. Conversely, subjecting peptide to extreme temperature wherehydrophobic collapse is kinetically favored over H-bonding may result inscaffolds having shorter fibril segments and more interfibrilcrosslinks. Resultant gels may have smaller pore sizes. Therefore,temperature control provides a general way to control nano- andmicroscopic features in the gel. In some embodiments, peptides may beassembled at two different temperatures (T₁=T_(gel) and T₂=T_(gel)+30°C.). Resultant hydrogels may be visualized via cryo-TEM and LSCM toassess nano- and microscale properties. Gels containing more interfibrilcrosslinks may be more rigid; this can be verified via rheology.

In some embodiments, peptides may be prepared that vary in sequencelength, for example, based on the general structure of MAX1, namelyVK(VK)_(m)VKVKV^(D)PPTKVKV(KV)_(n)KV-NH₂, where m=1-20 and n=1-20 and mmay be the same or different as n in any given peptide. Peptidescomposed of longer strand regions may form more rigid gels because ofthe increased hydrophobic surface area of longer hairpins. Peptidescomposed of longer strand regions that afford more rigid gels could beused to incorporated functional moieties. It has been shown thatmonomeric hairpins are most stable when their strands are approximately7 residues in length and lengthening them doesn't increase hairpinstability. Hydrogels of peptides having longer strand regions may beprepared and their rigidity assessed, for example, via rheology underidentical folding conditions.

In some embodiments, one or more amino acids of the turn region may besubstituted and/or modified as compared to the turn region of MAX1. Insome embodiments, turn sequences may be incorporated that not only playa structural role but also play a biofunctional role. For example, RGDbinding epitopes are normally found within turn regions of proteinsknown to be important in cell adhesion events, and residues that flankRGD provide additional specificity to the binding event. Incorporatingthese epitopes into the turn regions of self-assembling hairpins maylead to hydrogel scaffolds having enhanced cell adhesion properties. Asdiscussed above, incorporating a sequence that is incapable of formingturn structure results in a peptide (MAX9) that can not fold and doesnot form hydrogel. However, peptides that contain alternate turnsequences that are capable of adopting turn structure but are not aspromotive as the -V^(D)PPT- of MAX1 may still fold and undergohydrogelation. In the context of MAX1, peptides may be prepared in whichthe tetrapeptide sequence -V^(D)PPT- is replaced with sequences thatvary in their inherent propensity to form turn structure, from weak turnformers to strong turn formers. The ability of each peptide tointramolecularly fold and self-assemble may be determined by CD and therheological properties of their corresponding hydrogels may be studiedto identify hydrogels with desired characteristics (e.g., theirsuitability as possible scaffolds for tissue engineering). Examples ofpeptides that may be prepared include, but are not limited to,VKVKVKVK-XXXX-KVKVKVKV-NH₂ (SEQ ID NO:12), where -XXXX- is -V^(D)PGT-,very strong turn; -A^(D)PGT-, strong turn; -VNGT-, moderate turn; -VGGT-(SEQ ID NO:13), weak turn.

In some embodiments, functional moieties (e.g., cell adhesion epitopes)may be incorporated at one or more of: a) onto one or multiple sidechains of amino acids in the β-strand portion of the peptides, b) withinthe primary sequence at the ends and middle portions of the β-strands,c) at positions that flank the turn region, d) onto the side chain ofone or more of the amino acid residues forming the turn sequence, and/ore) within the primary sequence of the turn sequence. An example of afunctional moiety that may be used is the simple tripeptide RGD. Thesequences may be varied depending on the intended use of the hydrogel.For example, if the hydrogel is intended to be used as a scaffold forcells, the functional moiety may be varied depending on the knownsequences to elicit adhesion of specific cell types to be used. Examplesof specific adhesion sequences for various cell types are presentedbelow. Peptides in FIG. 19 may be prepared (side-chain modified peptideswill be synthesized using well established orthogonal protectionstrategies) and studied by CD and rheology to establish their foldingand rheological properties. Hydrogels prepared according to thisembodiment may have enhanced cell adhesion and proliferationcharacteristics.

In some embodiments, concentration of folded monomer present beforeself-assembly may be varied in order to produce a hydrogel havingdesired characteristics. CD may be used in concert with dynamic lightscattering (DLS) to determine a concentration of folded monomer thatmust be present in order for self-assembly to occur. DLS allows directmonitoring of nanostructure growth due to the self-assembly process.Distributions of particle sizes above 5 nm up to 1 micron can be readilyobserved. For example, using MAX1, CD may be used to monitor the rate ofβ-sheet formation at 20° C. The extent to which the magnitude of CDsignal can be attributed to intra- (due to folding) vs. intermolecular(due to self-assembly) β-sheet formation may be determined via areal-time comparison of the growth of CD β-sheet signal to the growth ofself-assembled particle size for identical solutions (μM peptideconcentrations). Importantly, folded monomers will not give rise to agrowth of particle size in DLS. Therefore, if a significant amount ofβ-sheet structure is observed by CD without concurrent particle sizegrowth then it can be concluded that a significant population of foldedmonomer must be present in order for self-assembly to occur. Conversely,if particle self-assembly occurs concurrently with CD β-sheet signalgrowth then one can conclude that self-assembly immediately followsintramolecular folding. A similar real-time comparison may be madebetween particle size growth in DLS and storage modulus growth monitoredvia rheology. Millimolar peptide concentrations will be used so thatdirect comparisons between DLS and rheology can be made. The thresholdof particle size above which network rheological properties areintroduced will be directly observed.

In some embodiments, hydrogels of the invention may exhibit an enhancedprocessibility. The processibility of the hydrogels of the invention maybe enhanced as compared to hydrogels known in the art as a result ofdesigning peptides having one or more active intramolecular foldingtriggers that allow peptide solutions to undergo hydrogelation on cue.As shown above, the ability to trigger folding provides a direct meansto control self-assembly and consequent hydrogel formation. In addition,a specific example of a suitable peptide secondary structure (i.e., thehairpin motif) has been provided, which is an ideal molecular scaffoldin which triggering elements can be easily incorporated. In someembodiments of the invention, hairpins can be designed to undergohydrogelation in response to physiologically relevant stimuli such aspH, ionic strength and temperature. In terms of processibility,triggering folding enables both in vitro and in vivo hydrogelationstrategies. In additional embodiments of the invention, additionalavenues may be used to trigger hydrogelation, for example,electromagnetic radiation (e.g., light) and specific ion binding (e.g.,calcium ion binding). Using electromagnetic radiation to triggerhydrogelation offers a convenient means to induce spatially resolvedmaterial formation in vivo via fiber optics. For example, low viscositypeptidic-cell suspensions can be administered and subsequently gelledlocally by irradiation. The ability to trigger hydrogelation ex vivo viastimuli naturally found in tissue provides another convenient means ofcontrolling material formation. Peptides may be prepared that undergohydrogelation in response to specific ion binding (e.g., calcium ionbinding). When calcium ion is used as a specific ion, resultantmaterials would not only serve as scaffolds upon which cells couldproliferate but also provide a source of calcium that would be releasedupon biodegradation of the peptide scaffold.

In the design of hydrogels of the invention, the folding andself-assembly process may be characterized by any suitable techniqueknown to those skilled in the art, for example, CD and FTIR. Materialnano- and microscale porosity may be determined via cryoTEM, LSCM,USANS, and SANS and material rigidity may be measured by rheology(using, for example, the methods described herein). The efficiency ofthe triggering process may also be examined. In some embodiments,triggering systems for peptides intended for in vivo hydrogelation maybe those affording fast hydrogelation leading to rigid, porous gels. Therate of trigger-induced sheet formation for dilute solutions of peptidemay be determined by CD and the rigidity of corresponding 1-2 wt %hydrogels may be determined by oscillatory rheology. For in vitroapplications, fast gelation kinetics may be desirable but aren'tnecessary. In a specific example, MAX1 may be used as a model system toinvestigate the effectiveness of each trigger discussed below. However,once the viability of a particular trigger has been established in thecontext of MAX1, the trigger can be incorporated into other peptidesequences that display desired attributes distinct from MAX1.

In some embodiments of the invention, electromagnetic radiation (e.g.,light) may be used to trigger the hydrogelation a peptide of theinvention. Examples of suitable methodology include those depicted FIG.20. The embodiments depicted in FIG. 20 include the use of photocages toinhibit folding. Such photocages may be incorporated in the peptides ofthe invention, for example, at the side chains of residues comprisingthe hairpin and/or within the peptide backbone. Folding and consequentself-assembly may be initiated by exposing aqueous solutions of peptideto light.

As discussed above, incorporating charged residues into the hydrophobicface of the hairpin (e.g. Glu) inhibits folding and consequentself-assembly. This inhibition may be utilized to inhibit folding viathe incorporation of the negatively charged photocage(2-nitrophenylacetic acid) on the hydrophobic face. In one embodiment,the amino acid residue at position 16 of MAX1 may be replaced with acysteine (MAX6) and may be alkylated with2-bromo-2-(2-nitrophenyl)acetic acid according to literature protocolaffording the caged peptide (Pan & Bayley, Febs Letters 1997, 405,81-85). 2-nitrophenylacetic acid is a commonly used photocage and itslight induced cleavage from heteroatoms has been thoroughly studied.Exposing caged peptides to light (330-360 nm) results in release of thenegatively charged cage and the generation of neutral Cys on thehydrophobic face. If the nitrosoketone by-product formed duringphotolysis is alkylating the newly formed Cys side chain (a potentiallydeleterious side-reaction), 1 mM dithiothreitol may be added to quenchthe nitrosoketone and maintain a reducing environment. Position 16 isinitially chosen for modification since a control peptide with Cys atthis position folds and self-assembles. One of ordinary skill in the artwill appreciate that one or more other positions in peptides may bytreated in a similar fashion. Photolysis efficiency may be dependent onsequence position and thus varying the location and/or number ofresidues photocaged may be used to vary the properties of the resultanthydrogels. In some embodiments, the rate of photolysis may be in themicro- to millisecond time regime. Typically, the period of irradiationmaybe from about a microsecond to several milliseconds, for example,from about 0.01 μs to about 1000 ms, from about 0.01 μs to about 100 ms,from about 0.01 μs to about 10 ms, from about 0.01 μs to about 1 ms,from about 0.01 μs to about 0.5 ms, from about 0.1 μs to about to about1000 ms, from about 0.1 μs to about 100 ms, from about 0.1 μs to about10 ms, from about 0.1 μs to about 1 ms, from about 0.1 μs to about 0.5ms, from about 1.0 μs to about 1000 ms, from about 1.0 μs to about 100ms, from about 1.0 μs to about 10 ms, from about 1.0 is to about 1 ms,from about 1.0 μs to about 0.5 ms, from about 10 μs to about 1000 ms,from about 10 μs to about 100 ms, from about 10 μis to about 10 ms, fromabout 10 μs to about 1 ms, or from about 10 μs to about 0.5 ms. Suitableirradiation sources include may provide bulk and/or spatially resolvedirradiation and may include, but are not limited to, a Nikon eclipseTE2000 inverted fluorescence microscope equipped with a mercury arc lampand UV laser to allow spatially resolved illumination; all commerciallyavailable.

In some embodiments, the N-alkylation of peptides may be used to inhibitH-bond driven self-assembly. β-hairpins that incorporate anN-o-nitrobenzyl cage at a backbone amide nitrogen that would normallyform an intramolecular H-bond (e.g. position 16 of MAX1) may besynthesized using standard techniques. A cage occupying this positionmay be used to inhibit intramolecular folding since intramolecularH-bonding is sterically hindered, FIG. 20. o-nitrobenzyl cages areanother class of compounds whose properties are well established.Exposure to light (330-360 μm) releases the cage resulting inregeneration of a protonated amide capable of engaging in H-bondformation and consequent folding/self-assembly. The efficiency ofphotolysis may varied dependent on sequence position and this may beused to vary formation and/or characteristics of the hydrogels. Sitespecific N-alkylated peptides may be prepared by solid phase peptidesynthesis (SPPS) via literature protocol (see Reichwein, et al.,Tetrahedron Letters 1998, 39, 1243-1246 and Tatsu, et al., Febs Letters2002, 525, 20-24). Since the particular cages described above have beenused in cellular context, cage by-products are unlikely to be cytotoxic.However, cytocompatibility of this system may be measured via methodsdescribed in the literature (see Bryant, et al., Journal of BiomaterialsScience-Polymer Edition 2000, 11, 439-457). Another possible concern isthat using light at 330-360 nm to trigger hydrogelation in the presenceof cells may lead to UV-based cellular photodamage since the o-nitroderivatives described above all involve one photon excitation to inducecleavage. Although one photon excitation has been commonly used in thecontext of cell-based experiments with no apparent damage to cells, ifUV-based damage is observed for any particular cell type, two photoncages that absorb in the IR (e.g. coumarin derivatives) may be used(Furuta, et al., Proceedings of the National Academy of Sciences of theUnited States of America 1999, 96, 1193-1200).

In some embodiments, binding of a specific ion may be use to triggerhydrogelation. For example, hydrogelation may be triggered via calciumion binding. In embodiments involving binding of calcium ion, typically,in the absence of calcium ion, peptides are unfolded while the additionof calcium nucleates folding/self-assembly. As shown above, the turnregion of the hairpin may be critical for promoting intramolecularfolding and consequent self-assembly. In one specific example, variantsof MAX1 may be prepared in which the four residue turn -V^(D)PPT- isreplaced with a turn/loop sequence or unnatural chelate incapable ofadopting ordered structure in the absence of metal ion. Metal bindingaffects turn formation which, in turn, promotes intramolecular foldingand self-assembly. FIG. 20 shows the metal-triggers described in detailbelow.

In one specific embodiment, a variant of MAX1 in which the four residueturn -V^(D)PPT- is replaced with -DRKADGYIDFEE- resulting in VKVKVKVKVDRKADGYIDFEE VKVKVKVKV (SEQ ID NO:14). This peptide should be unorderedin the absence of metal but rapidly fold and self-assemble after bindingCa(II). The sequence -DRKADGYIDFEE- corresponds to a calcium bindingloop contained within the EF hand domain of Troponin C. The underlinedresidues bind Ca(II) via both side chain and main chain carbonylsaffording a pentagonal bipyramidal geometry with water occupying one ofthe equatorial positions. Importantly, is has been shown that thislinear peptide is capable of binding Ca(II) with a low mM Kd and thatthe binding event leads to structure formation. The crystal structure oftroponin C indicates that the N- and C-terminal residues of the calciumbound loop are within about 6 Å and that these terminal residues adoptdihedral angles conducive to antiparallel sheet formation. In fact,energy minimization and dynamics simulations with explicit solvent wereperformed with this designed peptide and demonstrated that this metalbound loop is geometrically well suited to nucleate β-hairpin structurein the context of MAX1.

In another specific embodiment, an additional high affinity trigger maybe employed. This design invokes the use of a non-natural Ca(II)chelator DOTAM, which is a derivative of the thoroughly studiedoctadentate ligand DOTA, FIG. 20. DOTAM binds calcium tightly (Kd=29 nM)forming a square antiprism complex that situates the carbonyl containingarms of the ligand within 5 Å of each other, making these two positionsideal for peptide attachment. Therefore, replacing the four residue turn-V^(D)PPT- of MAX1 with the DOTAM-derivative in FIG. 20 affords a systemthat is unstructured in the absence of Ca(II); however, when Ca(II)binds, the two β-strands are brought within 5 Å of each other andfolding is initiated. This peptidomimetic may be prepared using manualsolid phase synthesis using commercially available or easily synthesizedstarting materials.

In some embodiments, peptide structure and material properties may beused to modulate the adhesion and proliferation of cell lines, forexample, stem cell, fibroblast and/or osteoblast cell lines. Hydrogelsdescribed above may be useful for tissue engineering applications (e.g.those that undergo triggered material formation via biocompatiblestimuli such as electrolyte concentration, temperature and lightaffording porous gels of sufficient rigidity to be self-supporting, >10Pa). The present disclosure will allow one of ordinary skill in the artto correlate peptide structure, material rigidity, and cytocompatibilityconcurrent with identifying hydrogels that may ultimately be useful intissue engineering. Three desirable cell level biologicalcharacteristics for tissue engineering feasibility are: a) the hydrogelsshould be noncytotoxic to desired cells b) the hydrogel should promotecell adhesion (attachment and spreading), and c) the hydrogel shouldallow cell proliferation. Hydrogels of the invention typically, althoughnot necessarily, possess one or more of these characteristics. First,the experimental strategy for assessing cytocompatibility (parts a, band c) will be described. Second, examples of cell lines suitable forthese studies will be discussed. Third, specific experiments aredescribed that will permit one of ordinary skill in the art to delineatethe interplay between peptide structure, material rigidity andcytocompatibility and to select appropriate peptides and triggers forparticular applications (e.g., particular cell types). In someembodiments, cell binding epitopes may be incorporated into the peptideto enhance material cytocompatibility. Examples of cell types that maybe used include, but are not limited to, mammalian cells, NIH 3T3fibroblasts, HEK 293, HELA cell lines, and rat calvarial MC3T3-E1osteoblasts, neuronal cells, stem cells, and cells derived from tissuesamples (e.g., brain, liver, heart, etc)).

In some embodiments, it is desirable to select peptides and triggeringconditions that facilitate cell adhesion and proliferation. Typically,four hours is sufficient time for fibroblast attachment, with almost allof the cells undergoing spreading but not proliferation. Literatureprotocol indicates that 12 hours is sufficient for osteoblast attachmentwithout proliferation. Therefore, fibroblast vitality may be assayedfour hours following their introduction to the hydrogel, for example, bypreferentially staining living versus dead cells and performingfluorescence-based imaging; osteoblasts may be assayed after 12 hours.Known numbers of respective cells may be seeded onto hydrogels preformedin tissue culture plates. After the respective time for each cell type,calcein AM or pentafluorobenzoyl aminofluorescein diacetate may be usedto stain the cytoplasm of living cells. Propidium iodide may be used tostain dead cells. Parallel controls using tissue culture plates withouthydrogel may be performed. If the number of dead cells in the hydrogelis higher than that observed in the control plates, then the materialmay be deemed unsuitable for the specific cell type. The possibilityexists that cells undergoing apoptosis may resist dye incorporationleading to an incorrect assessment of hydrogel cytotoxicity. Thecytotoxicity of these gels may be further verified in the adhesion andproliferation studies described below. Based on preliminary results, amajority of the proposed gels appear to be non-cytotoxic. Lastly, if theproposed dyes prove problematic, an alternate set of dyes may be used toassess cytotoxicity, for example, SYTO, a nucleic acid indicator of livecells used in combination with SYTOX, a nucleic acid indicator of deadcells.

In general, when synthetic materials are used in vitro as cell culturingsubstrates, cell adhesion is primarily mediated by serum proteins (e.g.fibronectin and vitronectrin) present in the growth media thatnon-specifically coat the scaffold. However, for hydrogels that mayultimately be introduced in vivo by the triggering mechanisms describedherein, the hydrogel scaffold may innately promote cell adhesion in theabsence of serum proteins. This ensures cell attachment when a potentialhost does not provide a mechanism to introduce cell adhesion proteins tothe scaffold. Hydrogels of the invention may promote cell adhesion bothin the presence and absence of 10% calf serum. An assay may be used toidentify appropriate hydrogels for cell attachment in which cellattachment is radiologically quantitated by determining the fraction ofattached versus unattached cells after gentle washing. Specifically, 1)a cell line of interest (e.g., mammalian stem cells, fibroblasts orosteoblasts) may be cultured and pre-labeled for 24 hours with ³Hthymidine; unincorporated label may be removed by washing and a two hourchase. 2) Known concentrations of labeled cells may then be introducedto both untreated tissue culture wells (control) and wells comprising ahydrogel of the invention. In wells containing hydrogel, cell growthmedia with or without calf serum may be added. 3) The kinetics of celladhesion may initially be monitored, for example, over six hours forfibroblasts in time increments that may be experimentally determined byone of ordinary skill in the art using routine experimentation. Thekinetics of osteoblast adhesion may initially be monitored over 24hours. These end time points may be experimentally refined; enough timemust be given to allow attachment and spreading but not proliferation.Also, a potential problem with end points that are too long is that somecells, if incubated for extended times, may modify the hydrogel surfacevia membrane bound proteases or secreted matrix proteins and allowattachment even on “non-stick” surfaces. In this scenario, attachment ispromoted by the cell and not necessarily the material surface. Optimumend points may be determined by plating cells onto surfaces coated withheat denaturated BSA (a typical non-stick surface). When greater than2-3% of the cells have attached, the maximal end point has been reached.4) At appropriate times, unattached cells may be gently washed away andcounted by liquid scintillation spectrometry and attached cells may besolubilized for scintillation counting employing stand curves forquantitation. Thus comparisons can be made among polystyrene tissueculture and hydrogel surfaces with and without serum proteins. Cellspreading following attachment may be optically verified. Using theinformation thus generated, a suitable hydrogel may be designed topromote the adhesion of any cell type of interest. By varying thepeptide structure, material properties such as rigidity and scaffoldmorphology may be adjusted to optimize a hydrogel's ability to fostercell adhesion. For example, simple cell spreading assays described aboveshowed that fibroblasts efficiently attached to MAX1 hydrogels (storagemodulus=1200 Pa) in the presence of serum proteins but less efficientlyin the absence of serum proteins. Hydrogels having this characteristicmay be useful for in vitro applications. By introducing structuralmodifications to the peptides and/or by preparing hydrogels of differingrigidity enhanced cell adhesion in the absence of serum proteins may beobtained. Hydrogels with enhanced cell adhesion may be used for both invitro and in vivo applications and may be more suited for in vivoapplications than hydrogels having a lower degree of cell adhesion.

In some embodiments, non-cytotoxic hydrogels that promote cell adhesionas outlined above may be used to support the proliferation of theadhered cells. Proliferation rates may be assessed using the ³Hthymidine assay described above. For a given gel, the optimal cellseeding density may be determined, for example, by using rateexperiments lasting 48 hours for fibroblasts. With reference to FIG. 17,initially seeding of MAX1 hydrogels with 40,000 fibroblasts allowsproliferation to be followed for 2 days until confluency is reached.Proliferation may be followed for any suitable length of time, forexample, over two days or over longer time periods. The duration of rateexperiments involving other cell types (e.g., osteoblasts, neuronalcells, stem cells, etc.) may be experimentally determined in a similarmanner. After the optimal seeding density has been determined, quiescentcells may be seeded onto hydrogel and the number of proliferating cellsmay be quantitated as a function of time via ³H thymidine uptake. In oneembodiment, cells at optimal density may be plated onto preformedhydrogels in 24 well tissue culture plates in the presence of unlabeledmedia containing 10% calf serum. In triplicate, cells may be allowed toproliferate for distinct time periods (e.g. 6, 12, 18 hours, etc.).After each discrete time, growth media may be removed and replaced withmedia containing ³H thymidine. Cells may be allowed to uptake ³Hthymidine for three hours. Unincorporated thymidine may be washed out(washes may be assayed until ³H thymidine is no longer detected) and thecells may be sacrificed for scintillation counting. Comparing tostandard curves (which may be previously determined by culturing desiredcells on tissue culture polystyrene and directly correlating cellscounted by hemocytometry to scintillation counts) allows one to quantifycells undergoing DNA replication at that time. In some embodiments, thenumber of proliferating cells on certain hydrogels may exceed the numberthat could possibly fit within the two dimensional area of the hydrogelsurface. In these cases, cells may have proliferated into the pores andchannels of the hydrogel scaffold. In some embodiments, hydrogels of theinvention may be designed to promote the proliferation of cells into thehydrogel scaffold. Hydrogels of this type may be readily identified, forexample, by following the proliferation of pre-stained cells via LSCMwhere 3-D images may be collected as a function of time; cells that havemigrated into the hydrogel interior will be clearly visible. Cells maybe pre-stained with any suitable dye, for example, Vybrant CFDA SE(Molecular Probes), a green fluorescent probe that is sequestered in thecytoplasm of cells and is passed on to daughter cells (cell viability isunaffected). One of ordinary skill in the art may use assays of thistype to identify hydrogels having the desired ability to promote cellproliferation. By varying peptide structure and the resultant materialproperties of the hydrogel such as rigidity and scaffold morphology,hydrogels may be produce that foster cell proliferation as discussedbelow.

The assays described above will be useful in assessing adhesion andproliferation on the surface of two dimensional hydrogels and allowfacile comparison among different hydrogel scaffolds, and the resultscan be directly compared to materials in the literature since mostpublished work has been performed in 2-D. As shown in FIG. 18,triggering material formation in the presence of cells allows thethree-dimensional incorporation of these cells throughout the hydrogel.Non-cytotoxic hydrogels that promote cell adhesion (for example,hydrogels identified as above) may also be used as 3-D substrates. Cellattachment may be difficult to quantify since unattached cellssequestered in the interior of hydrogel can not be washed away. However,a qualitative cell spreading assay may be used to gauge cell spreadingand thus attachment. Cells may be pre-stained, for example, with VybrantCFDA SE and subsequently incorporated into the hydrogel matrix. Cellmorphology of initially incorporated cells may be visualized in threedimensions by LSCM (non adhered cells will appear round and adheredcells will appear spread). In addition, since the dye is passed on toprogeny, cell proliferation can be qualitatively visualized.Proliferation rates can be quantitatively determined using the sameassay as described above for the 2-D work. ³H thymidine diffuses readilyinto the hydrogel matrix and should be available to cells undergoing DNAreplication.

In some embodiments, the present invention may be used to support theadhesion and/or proliferation of cells (e.g., cell lines). Such cells orcell lines may be of any type known to those skilled in the art and maybe cultivated for any purpose (e.g., to generate a tissue or forexpression of a desired protein). In embodiments related to tissueengineering, model cell lines may be chosen based upon theirrelationship to human cells involved in connective tissue and bonegeneration/healing in addition to their robust performance during invitro culturing. Examples of suitable cell lines for this purposeinclude, but are not limited to NIH/3T3 mouse fibroblasts, animmortalized cell line, which may be used as a model of fibroblastsinvolved in human connective tissue development and rat calvarial(MC3T3-E1 Subclone 4) cells, which may be used as a model of humanosteoblast cells. Both cell lines are well studied and a significantliterature database exists allowing the performance of the proposedhydrogels to be benchmarked against previously studied materials. One ofordinary skill in the art is aware of the possible limitations using theMC3T3-E1 cell line such as their ability to revert to anon-differentiating fibroblast-like phenotype. In some embodiments,hydrogels of the invention may be used to support the adhesion and/orproliferation of cells derived from primary tissue samples, for example,primary osteoblasts from calvaria or bone marrow.

In some embodiments, the hydrogels of the invention, which may containone or more cells and/or one or more therapeutic agents (e.g.,pharmaceuticals). Hydrogels for this purpose may be identified usingstandard techniques in animal model systems. Hydrogels may be preformedprior to insertion into an animal or may be inserted into in the form ofa solution and undergo triggered hydrogelation in situ. In someembodiments, hydrogels may be formed by peptides that undergo triggeredmaterial formation via biologically relevant stimuli. For example, asolution of peptides, which may include cells and/or therapeutic agents,may be inserted into an animal (e.g., a human) and undergo hydrogelationas a result of the one or more triggers endogenous to the animal (e.g.,pH, ionic strength, etc). In other embodiments, hydrogelation may beeffected using an exogenous trigger, for example, electromagneticradiation (e.g., light). Using materials and methods of the invention,scaffolds with desired rigidity may be constructed and used in vivo.Such scaffolds may provide a substrate with optimal mechanicalproperties for adhesion and proliferation of cells and/or delivery oftherapeutics.

In some embodiments, the present invention provides a method forpredicting the cytocompatibility of a hydrogel. For example, theexperimental data from the cytocompatibility assays described above maybe used to draw correlations between peptide structure presented in thestudied hydrogels and cytocompatibility. In some embodiments,correlations between cytocompatibility and molecular parameters such aspeptide hydrophobicity, β-hairpin strand length, and other parametersdiscussed above may be identified and used to predict thecytocompatibility of proposed hydrogel. The molecular parameters mayhave been varied in order to understand and control the self-assemblyprocess. These correlations will then allow the design of completely newpeptides with both optimal material and cytocompatibility properties inan iterative design process.

In a specific embodiment, cell adhesion epitopes may be incorporatedinto hairpin-based hydrogels in order to enhance cell attachment in theabsence of serum proteins. Enhancement in the rate of proliferation mayalso be measured. The two-dimensional quantitative cell attachment andproliferation assays previously described may be used. In oneembodiment, cell adhesion epitopes may be incorporated into hydrogels.One of ordinary skill in the art will appreciate that there is clearexperimental evidence that the addition of cell adhesion epitopes withintissue engineering matrices promotes cell attachment and/orproliferation (see, for example, Urry, Angewandte Chemie-InternationalEdition in English 1993, 32, 819-841, David, et al., BioconjugateChemistry 2001, 12, 890-899, Rezania & Healy, Journal of OrthopaedicResearch 1999, 17, 615-623, Cook, et al., Journal of BiomedicalMaterials Research 1997, 35, 513-523, Burdick & Anseth, Biomaterials2002, 23, 4315-4323, Houseman & Mrksich, Biomaterials 2001, 22, 943-955,Kao, et al., Journal of Biomedical Materials Research 2001, 55, 79-88,and Schmedlen, et al., Biomaterials 2002, 23, 4325-4332). One or morecell binding ligands may be covalently attached to individual hairpinsand self-assembled into a hydrogel matrix. One or more sequence epitopesmay be incorporated at any position of the peptide, for example, at oneor multiple lysine side chains or within the primary sequence of apeptide (e.g., MAX1). Any position that can tolerate the additionalepitopes without adversely affecting the self-assembly as discussedabove may be used. Within the structural context of MAX1, one canincorporate one or multiple epitopes allowing precise control of theconcentration of epitopes displayed. In addition, copies of epitopes canbe identical or can vary in identity. For example, synergistic ligandbinding sites are known to exist in α5β1; mutagenesis experiments onfibronectin demonstrate that RGD is a more effective ligand withneighboring SDV and RNS epitopes (Ruoslahti, Annual Review of Cell andDevelopmental Biology 1996, 12, 697-715). Therefore, in someembodiments, hairpins rich in epitope diversity may be prepared and usedto promote cell adhesion and proliferation. In some embodiments, one ormore integrin binding ligands may be incorporated. Such integrin bindingligands may be based on extracellular matrix proteins specific tofibroblasts and epitopes from bone sialoprotein specific to osteoblasts.The epitopes to be incorporated may be selected to be complementary tothe cell line to be used with the hydrogel.

In one specific embodiment, a hydrogel suitable for use with 3T3fibroblasts may be prepared. Such a hydrogel may incorporate one or moreRGD epitopes known to bind α5β1. This integrin has been shown to existin fibroblasts cultured from healthy human periodontal connectivetissues (Hakkinen, et al., Biochimica Et Biophysica Acta-Molecular CellResearch 1994, 1224, 33-42). In specific embodiments, one or more of thepeptide epitopes TRGDSP (SEQ ID NO:15), RGDG (SEQ ID NO:16), RGDY (SEQID NO:17), and RGDW (SEQ ID NO:18) may be incorporated into the peptidesat any position and used to prepare a hydrogel of the invention. One ormore copies of each epitope may be incorporated alone or in combinationwith one or more copies of the other epitopes.

In another specific embodiment, a hydrogel suitable for use with MC3T3osteoblasts may be prepared. Such a hydrogel may incorporate one or morecopies of the epitope FHRRIKA (SEQ ID NO:19), an epitope derived frombone sialoprotein, known to bind to heparan sulfate proteoglycansdisplayed on osteoblast surfaces. In other embodiments, hydrogels of theinvention may incorporate an additional epitope known to positivelyaffect osteoblast adhesion, GRGDSPY (SEQ ID NO:20). Hydrogels may beprepared with one or more copy of either epitope or with one or morecopies of both epitopes in combination. Rezania & Healy (supra) haveobserved an increase in osteoblast proliferation when RGD-containingpeptides and FHRRIKA were used together.

In some embodiments, suitable epitopes for enhancing cell binding and/orproliferation may be identified using competitive binding assays withsoluble epitope. For example, if cell attachment and/or proliferation ismediated by specific ligand binding events, enhancement of attachment orproliferation should be competitively inhibited by increasingconcentrations of added soluble ligand. In addition, for those peptidescontaining RGD sequences that lead to significant enhancements, controlsequences will be prepared in which the 1-aspartic acid residue in theRGD sequence will be replaced with d-aspartic acid. It has been shownthat this simple change in stereochemistry is completely deleterious tointegrin binding.

The present invention also encompasses hydrogels having varyingstiffness. Varying the stiffness of the gel can be used to modulatecell-material interactions, specifically cell adhesion andproliferation. Hydrogels of varying stiffness may be prepared whilekeeping the peptide primary sequence constant. In one specificembodiment, the peptide MAX1 may be used to prepare hydrogels of varyingstiffness. The rigidity of MAX1 hydrogels may be varied by alteringpeptide folding triggering conditions, namely folding temperature, whileholding peptide concentration constant. Based on preliminary dataaccessible storage moduli (gel stiffness) will range from several Pa to10,000 Pa. MAX1 gels formed at different temperatures retain theirrigidity even after being re-equilibrated at 37° C. (e.g., cellculturing conditions). By subsequently observing two-dimensional celladhesion and proliferation on hydrogels of varying stiffness, theoptimal substrate stiffness for any particular cell type (e.g.,neuronal-material, stem cells, fibroblast-material and/orosteoblast-material) may be determined. Although MAX1 may be used forthis purpose, the stiffness of a hydrogel may be varied using anypeptide described herein. Varying the stiffness of the gel may also beused to optimize the cytocompatibility of the hydrogels.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein are obvious and may be made withoutdeparting from the scope of the invention or any embodiment thereof.Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

EXAMPLE 1

Intramolecular folding events, triggered by the presence of salt, inducethe self-assembly of β-hairpin peptides into hydrogel networks atphysiological conditions. At pH 7.4, low ionic strength solutionconditions, dilute, homogeneous solutions of peptide (≦2 wt. %) exhibitthe viscosity of pure water. Circular dichroism spectroscopy shows thatat pH 7.4 in the absence of salt, peptides are unfolded. By raising theionic strength of solution, electrostatic interactions between chargedamino acids within the peptide are screened and a β-hairpin conformationis adopted. Folded β-hairpin molecules supramolecularly assemble viahydrophobic collapse and hydrogen bonding into a three dimensionalhydrogel network. FTIR and x-ray scattering data demonstrate that thesehydrogels are rich in β-sheet. Dynamic oscillatory rheologicalmeasurements demonstrate that the resultant supramolecular structureforms an elastic material whose structure, and thus modulus, can betuned by salt concentration and temperature. Storage moduli of hydrogelsincrease with increasing salt concentration. Robust hydrogelation isalso observed in cell growth media at physiological conditions.Transmission electron microscopy reveals that the hydrogel elasticityarises from a network nanostructure consisting of semi-flexiblefibrillar assemblies.

Hydrogels are collectively an important class of biomaterial that haveextensive uses in tissue engineering and drug delivery applications.Self-assembly strategies provide precise control in order to constructnew hydrogel materials with desired nano- and micro-structures that canbe responsive to environmental conditions, such as temperature, pH,electric field, ionic strength or light. Hydrogelation of β-hairpinpeptide using pH and temperature triggers has been shown (see Pochan2003 and Schneider 2002 supra). These molecules exhibited a pH andtemperature dependent intramolecular folding event that resulted in areversible intermolecular self-assembly process leading to hydrogelscaffold formation. In general, by controlling the peptide-foldingevent, it is possible to design responsive materials that undergoself-assembly with desired stimuli. In addition, the self-assemblyprocess can be controlled in order to engineer desired morphological andmechanical properties. In one specific embodiment shown below, saltconcentration is used as a trigger to induce the β-hairpinintramolecular folding event followed by self-assembly into asupramolecular elastic network. The rate of folding, and thus, final gelrheological properties, may be tuned by ionic strength of the peptidesolutions. Importantly, the formation of these physical hydrogels doesnot involve any chemical crosslinking chemistry and can be performed atphysiological temperature and pH.

There are different strategies reported in the literature to makepolymeric gels that are responsive to stimuli, e.g. undergo sol-gel orswelling transitions as a response to changes in physical or chemicalenvironmental conditions. While temperature and pH are the mostprevalent stimuli, ionic strength is also used to trigger gelation byphase transition, self-assembly or polymer conformational changes. Theformation of salt complexes can change solution and self-assemblyproperties of block copolymers that form self-assembled structures likespherical or worm-like micelles. For example, the type and amount ofsalt has been shown to change aggregation properties and, thus, theelasticity of the networks formed by Pluronic based block copolymersystems. Chemical hydrogels of synthetic polymers such asN-isopropylacrylamide, well known to exhibit thermal responsiveness dueto an LCST-type phase transition, also exhibit salt induced volume phasetransitions. At relatively high salt concentrations (or at temperatureshigher than the LCST), hydrophobic interactions dominate and lead to theprecipitation of the chemically crosslinked polymer chains causing gelcollapse. Swelling of hydrophilic networks such as poly(acrylic acid)and poly(methacrylic acid) hydrogels caused by ionic strength and pH hasbeen explored for the potential to produce responsive biomaterials.

Gelation behavior of biomacromolecules, such as gelatin,polysaccharides, and β-lactoglobulin, is extensively studied in theliterature. It is well known that by applying thermal treatments, thesemacromolecules undergo secondary structure (conformational) phasetransitions, resulting in the formation of intermolecular networkstructures. For example, gelatin forms hydrogels with decreasingtemperature via the formation of triple helical physical junctions.Rheological studies on gelatin gels revealed that the viscoelasticproperties are highly dependent on processing conditions, such as rateof cooling, degree of undercooling and concentration. However, due tothe polyelectrolyetic nature of biomacromolecule solutions, ionicstrength is also a vital parameter to control assembly properties.Effects of ionic strength on biomacromolecular gel networks, such aspectin, β-lactoglobulin, welan, and gelatin have been well studied dueto their importance in food processing and pharmaceutical applications.In general, increasing the ionic strength of these solutions results inan increase in network elasticity due to the formation of intermolecularsalt-bridge physical crosslinks.

There is a great interest in mimicking the stimuli responsiveself-assembly routes of biomacromolecules with well-defined sequences ofoligo and poly-peptides because of their potential applications asresponsive biomaterials. Self-assembly of oligopeptides into i-sheetrich, β-amyloid-like structures triggered by ionic strength and pH hasbeen investigated as a biomimetic material formation strategy. Forexample, Caplan et al. (Biomacromolecules 2000, 1, 627-631) showed thatself-assembly and resultant gel properties of an oligopeptide, with analternating sequence of hydrophobic amino acid residues with positivelyor negatively charged amino acids, can be altered by the ionic strengthof the solution. In addition, elastin-mimetic peptides (McMillan andConticello, Macromolecules 2000, 33, 4809-4821), hybrid molecules withcoiled-coil protein-synthetic polymer domains (Wang, et al., Nature1999, 397, 417-420), and leucine zipper polypeptide domains withadditional polyelectrolyte segment (Petka et al. Science 1998, 281,389-392), form responsive hydrogels that undergo gelation withtemperature or pH at appropriate ionic strength.

In the specific embodiment detailed below, salt-triggered physicalhydrogel formation via intramolecular folding and consequentintermolecular self-assembly of a 20 amino acid β-hairpin molecule(MAX1) at low peptide concentrations (2 wt %) under physiologicalconditions is demonstrated. The molecule consists of two strandscomposed of an alternating sequence of valine (V), (isopropylhydrocarbon side chain) and lysine (K) (primary amine capped side chain)amino acid residues that are connected with a tetrapeptide turn sequence(-V^(D)PPT-) that adopts what is known as a type II′ turn. While V is anonpolar residue, K is positively charged and hydrophilic at pH valuesaround physiological conditions (˜7.4). Electrostatic forces andhydrophobic interactions, due to the charges on K residues and nonpolarV residues respectively, are the primary intramolecular parameters thatcan be used to control the folding of the molecule. In the folded statethe molecule is facially amphiphilic, having all V residues on one faceof the hairpin with K residues on the other. Once the molecule is foldedself-assembly is driven by both lateral intramolecular hydrogen bondingand facial hydrophobic interactions. Therefore, these molecules arespecifically designed to first fold, and then self-assemble intoquaternary structures rich in β-sheet that gives rise to gel propertiesof the system. Final elastic properties of the self-assembled structurecan be tuned by salt concentration. In addition, due to the facialhydrophobic association, temperature can also be used as a parameter incontrolling gelation kinetics. Therefore, these molecules are designedto exhibit sol-gel transitions when solution conditions (ionic strength,temperature, pH) are adjusted to physiological levels providingsignificant potential uses for tissue engineering applications.

Sample Preparation: MAX1 was prepared on amide resin via automated Fmocbased solid phase peptide synthesis employing an ABI 433A peptidesynthesizer and HBTU/HOBT activation. The details of peptide preparationare given elsewhere (Schneider supra). Hydrogels are prepared bydissolving the lyophilized peptide first in DI water and the desiredfinal solution conditions achieved by the subsequent addition of bufferand salt containing solution. Bis-trispropane is used for buffering thesolution at pH 7.4 with the exception of the x-ray study in which TRISbuffer was used.

Circular Dichroism: CD spectra were collected using an Aviv model 215spectropolarimeter. Measurements were done either at 20° C. or 37° C.Wavelength scans, between 190 and 260 nm, for 2 wt % MAX1 (pH 7.4, 50 mMBTP) with 0 mM and 150 mM KF solutions were obtained in a 0.01 mmdetachable quartz cell. The spectra were taken after 2 hours ofdissolution of peptide in DI water and buffer solution. For timedependent studies, mean residue ellipticity θ, was measured at 218 nm. θwas calculated from the equation θ=θ_(obs)/l/c/r, where l is the pathlength of the cell, c is the concentration and r is the number ofresidues.

Infrared Spectroscopy: IR spectra were taken on a Nicolet Magna-IR 860spectrometer. Deuterated MAX1*nDCl was prepared by lyophilizing the TFAsalt of peptide once from 0.1 M HCl and twice from D₂O. Samples werekept in a temperature controlled water bath at 20° C. for two hoursbefore measurements were immediately taken at room temperature in azinc-selenide flow cell with 30 μm path length. The equipment wasoperated at 1 cm⁻¹ resolution and the spectrum recorded was an averageof 100 scans.

Wide Angle X-Ray Scattering: X-Ray spectra of the hydrated gels werecollected at the National Synchrotron Light Source, Brookhaven NationalLaboratory, beamline, X10A. Hydrogel was smeared on Kapton tape justbefore taking measurements in order to avoid dehydration. Data wascollected for 10 minutes on a two dimensional Bruker CCD array. Peptidesolutions were buffered with 125 mM TRIS. Measurements were done at roomtemperature.

Rheology: Dynamic time and frequency sweep experiments were performed ina strain controlled Rheometrics ARES rheometer with 25.0 mm diameterparallel plate geometry and 0.5 mm gap distance. Lyophilized peptide wasdissolved in de-ionized water and buffer or cell growth media solutionat 10° C. in order to suppress folding and gelation before the sample isloaded into the rheometer. After the sample was loaded, temperature wasincreased to either 20° C. or 37° C. Standard low viscosity mineral oilwas used to insulate the sides of the plate in order to suppressevaporation. Control experiments showed that mineral oil had no effecton rheological measurements. Strain sweep experiments were performed todetermine the linear viscoelastic regime in which all rheologicalexperiments were performed. Dynamic time sweep experiments wereperformed with a frequency and strain of 6 rad/s and 1%, respectively.Frequency sweep experiments were performed with a frequency and strainof 0.1-100 rad/s and %, respectively.

Transmission Electron Microscopy: A very thin layer of hydrogel wasapplied to carbon coated copper grids. The samples were negativelystained by placing a drop of 2 wt/v % of uranyl acetate aqueous solutionon the grid. The excess of the solution was blotted with filter paperand the sample subsequently left to dry. To prevent the formation ofsalt and/or buffer crystals, and to image the individual fibrilsclearly, hydrogel with 2 wt % peptide concentration was diluted to ˜0.1wt % in DI water. To disperse the fibrils evenly in the solution, gentlesonication was employed with a tip sonicator. Bright field images ofhydrogel nanostructure were taken on a JEOL 2000-FX transmissionelectron microscope at 200 kV accelerating voltage on both a Gatan CCDcamera and Kodak negative films.

FIG. 21A shows CD spectra of 2 wt % MAX1 solutions at pH 7.4 and 20° C.for different salt concentrations. Both spectra shown were taken after 2h of dissolution of MAX1 with DI water and buffer solution. The minimaat 218 nm for the peptide solution with 150 mM KF indicates that at 2 wt% gelation concentrations MAX1 folds and adopts secondary structure richin β-sheet. However, 2 wt % MAX1 at identical pH in the absence of saltdid not show significant regular secondary structure even after 2 hr,indicating the peptide remains unfolded without the presence of addedsalt.

Folding of the peptide molecule to β-hairpin structure and subsequentβ-Sheet formation with the presence of salt was also confirmed by FTIRspectroscopy. The FTIR spectrum of MAX1 solution at pH 7.4 and with 400mM NaCl, between 1580 and 1700 cm⁻¹, is given in FIG. 21B. The amide Iband for unordered peptide in D₂O is centered around 1645 cm⁻¹. Theshift of the amide I band from 1644 cm⁻¹ to 1614 cm⁻¹ suggests that thepeptide is in β-sheet conformation in 400 mM NaCl solution. The weakband in the spectrum at 1680 cm⁻¹ may be an indication of antiparallelβ-sheet structure. For MAX1 solution without NaCl, a very broad spectrumis obtained in the plotted range. The strong band at 1644 cm⁻¹ suggeststhat peptide is in an unordered state. However, the weak band at 1614cm⁻¹ also indicates the presence of a small amount of folded β-hairpinmolecules in the solution. We believe that folding of some of MAX1 isdue both to the excess buffer salt and relatively high concentration ofpeptide in the solution. It was shown that peptide concentration has aneffect on the kinetics of β-sheet formation. Therefore, this smallamount of β-sheet amide I signal, in the context of the highconcentration of peptide and lack of gelation is insignificant.

Since MAX1 has a net positive charge at pH 7.4 due to the primary amineson the lysine residues the data suggest that it cannot fold due to theelectrostatic repulsion between the strands. Therefore, when thepositive charges on the lysine residues are screened by Cl⁻ ions theintramolecular folding event is favored leading to formation ofβ-hairpin structure. Increased salt concentrations may also drive thehydrophobic association of residues, which should also contribute tofolding and self-assembly. Both CD and FTIR data show that the foldingmechanism is triggered by ionic strength of the solution. It has beenshown that folding can be triggered by pH and temperature (Pochan,Schneider supra). Similar to the effect of salt, when pH is used as astimulus, lysine residues are deprotonated at high pH and MAX1 folds.When folded, MAX1 is capable of self-assembly into higher orderstructures by intermolecular hydrogen bonding and hydrophobicinteractions. The kinetics of folding, structure of assembledaggregates, and the consequent hydrogel network properties will bediscussed below.

Crystallographic β-sheet structure is observed in the final hydrogelsusing x-ray scattering techniques. FIG. 21C shows the wide-angle curvesfor a hydrogel consisting of 3 wt % of MAX1 and 150 mM NaCl. Thescattering peak corresponds to 4.7 Å. This is the characteristicsignature of the interchain distance in β-sheet rich structures. Sincethe measurements were taken without drying out the hydrogels, thescattering background contribution at high angles due to the waterstructure is significant.

Hydrogels formed via the intramolecular folding mechanism exhibit rigidviscoelastic properties, even at low concentration of MAX1 peptide, asobserved by dynamic oscillatory techniques. FIG. 22A shows frequencysweep measurements from 10⁻¹ to 10² rad/s for 2 wt % MAX1 peptidesolutions at 20, 150 and 400 mM NaCl concentrations. Before thefrequency sweep measurements peptide solutions were allowed to gel for2.5 hr in the rheometer (FIG. 22B). For MAX1 solution with 400 mM NaClthe equilibrium storage modulus (G′) is ˜3000 Pa. Decreasing saltconcentration resulted in the decrease of both G′ and loss modulus (G″)values. A hydrogel with low rigidity (G′ ˜100 Pa) is formed when only 20mM NaCl is present in the solution. MAX1 at pH 7.4 without added NaCldid not exhibit any gelation even after 3 hours with G′ valuesapproximately 1 Pa (the response of the sample to the applied strain wasnegligible; torque values were under the detection limit of theinstrument leading to insignificant measurements that are not shown).For all three samples with salt, G′ values are at least one order ofmagnitude greater than the G″ values. Also, for all NaCl concentrations,the G′ values are essentially independent of the frequency in thestudied range exhibiting no crossover point between G′ and G″ at lowfrequencies. These characteristics are a clear signature of crosslinkednetworks. The frequency sweep data in FIG. 22A suggests that thesesolutions form rigid, solid-like hydrogels with properties similar tocovalently cross-linked polymer gels.

The spectroscopy data in FIG. 21 suggests that in solutions without saltthe electrostatic repulsion of lysine residues keeps the peptideunfolded, thus preventing self-assembly into a network structure.However, once the molecule is folded, self-assembly can occur. Since allvaline residues are positioned on one side of the molecule in the foldedstate, facial hydrophobic dimerization can occur. Additionalintermolecular hydrophobic interactions and hydrogen bonding canconsequently occur forming an interconnected network. Importantly, thedifferences in the hydrogel rigidity with different salt concentrations,as shown in FIG. 22A, reveal that not only can the self-assembly betriggered with ionic strength, but also that the resultant nanostructurecan be predictably altered by the ionic strength of the medium. FIG. 22Bshows the increase in G′ values during self-assembly of MAX1 solutionfor the same peptide concentration of 2 wt %. This experiment clearlydemonstrates the differences in the kinetics of self-assembly andnetwork formation for different salt concentrations. MAX1 solution with400 mM NaCl shows a rapid increase in its G′ value in the first coupleof minutes of the self-assembly process while the 20 and 150 mM NaClsolutions stiffened with a relatively slow rate. In fact, the 20 mM NaClsolution G′ values do not change significantly for 3000 seconds,remaining around 2-3 Pa. This lag time is shorter for 150 mM (˜1000 sec)and does not exist for 400 mM salt solution. FIG. 22B clearly suggeststhat faster kinetics of folding and self-assembly due to higher ionicstrength results in stiffer gels. The order of magnitude differences instiffness between solutions with the same peptide concentrationpresumably arise from more highly crosslinked networks due to fasterfolding and assembly kinetics.

The nanostructure of the hydrogels that results in elastic propertieswas studied by transmission electron microscopy (TEM). FIG. 23A showsthe TEM image of a 2 wt % MAX1 hydrogel network formed at pH 7.4 with400 mM NaCl. The micrograph reveals the highly interconnected fibrillarnetwork structure at the nanoscale. Although it cannot be directly seenfrom the TEM micrograph, it is believed that most of the junction pointsformed during self-assembly are intersecting fibrils and are not simplyentanglements of long, nonintersecting fibrils (although certainlyentanglements contribute to the modulus). These crosslink points betweenthe fibrils, although not covalent in origin, are permanent and areformed via both hydrogen bonding and hydrophobic interactions. Frequencysweep data, given in FIG. 22A, also supports this view; G′ isinsensitive to frequency, is an order of magnitude greater than G″, andno G″ to G′ crossover exists. Due to the evaporation of the water priorto imaging and relatively high salt concentration the 2 wt % structureis very dense with some parts of the network embedded in precipitatedsalt. Therefore, the hydrogel was diluted with DI water by approximatelya factor of 20 (to ˜0.1 wt % peptide) and a sample was immediatelyprepared for TEM imaging. FIG. 23B shows the more dilute fibrillarassemblies. Contour lengths of the fibrils are on the order ofmicrometers. Although drying during sample preparation may causeconformational changes along the fibril axis, micrographs suggest thatthe self-assembled fibrils are semi-flexible. Importantly, the widths ofthe fibrils are monodisperse in size and approximately 3 nm. Theproposed local self-assembled structure and the dimensions are shown inFIG. 24. Insight II modeling shows that the strand axis of the foldedpeptide is 32 Å in length and the distance from valine face to lysineface is ˜10 Å. The ˜3 nm width of the fibrils is in very good agreementwith these molecular dimensions. TEM data along with the CD datastrongly suggests that MAX1 is in the folded state during theself-assembly process. In the proposed structure in FIG. 24, β-turns areshown to be on the same side of the fibrillar bilayer for simplicity.

CD and rheology results clearly demonstrate that folding and subsequentgelation of β-hairpin molecules can be triggered with a change in saltconcentration at pH 7.4. This type of response provides an opportunityfor using these materials at physiological conditions. Therefore, theself-assembly and gelation behavior of these molecules was studied atphysiological temperature, 37° C. It has been shown that folding andβ-sheet formation of MAX1 is temperature dependent. CD studies indicatedthat for MAX1 the transition temperature from an unordered, random coilstate to folded β-sheet structure is around 25° C. at pH 9 and low (20mM) salt. This transition was shown to be tunable to higher temperaturesvia changing the hydrophobic character of the assembling peptide.

To observe the effect of temperature on the self-assembly triggered byionic strength, gelation was monitored over 2.5 hours by dynamic timesweep experiments at 20° C. and 37° C. In FIG. 25A, the change in G′ isplotted as a function of time for 2 wt % MAX1 at pH 7.4 and 150 mM NaClsolution. In both cases MAX1 solutions were initially kept at 10° C. tosuppress folding before measurements were taken. An instantaneousincrease in G′ at 37° C. indicates that the peptide formed a networkstructure immediately during self-assembly. Contrastingly, at 20° C. theincrease in elastic response of the hydrogel is slower showing aninsignificant increase in G′ for the first 15 minutes of self-assembly.This behavior can be beneficial for potential applications in which onedesires a solution with viscous-like behavior at room temperature whileexhibiting a fast gelation response when exposed (e.g. injected in vivo)to body temperature and salt concentration. FIG. 25A clearly shows thatat physiologically relevant conditions (e.g. pH 7.4, 37° C.), gelationleading to a material that displays useful rigidity occurs in about 10minutes. The frequency sweep measurement (FIG. 25B) taken at the end of2.5 hours gelation period at 37° C., indicates that a rigid hydrogel(G′>G″) was formed with a G′value around 2000 Pa. During gelation atboth temperatures G″ values (not shown) were constant and always wellbelow (>1 order of magnitude) the G′ values throughout the gelationprocess. In all samples, even during the early stages of gelation,G′>G″. Similar behavior has also been seen in the gelation ofbiomacromolecules. Similar to ionic strength effect, the formation ofstiffer gels assembled at higher temperatures may be due to structuraldifferences at the nanoscale. A faster rate of folding and consequentself-assembly can form network structure more dense in number ofjunction points leading to higher G′ values. This data shows that therigidity of MAX1 hydrogel can be easily and predictably controlled byeither temperature or salt concentration.

The rate of folding and β-sheet formation of MAX1 peptide was studied byCD. FIG. 25C shows the rate of change of ellipticity, measured at 218nm, at 20 and 37° C. for 2.0 wt % MAX1, pH 7.4 with 150 mM KF solution.KF is used as the electrolyte since it is optically silent and conduciveto CD measurements. CD data reveals that in the early stages ofself-assembly, β-sheet formation is very fast at 37° C. The rate offolding during the first 5 minutes is significantly high, reaching aplateau region after 15 minutes. However, at 20° C. the rate of foldingis slower. The differences in the kinetics of β-sheet formation and,thus, self-assembly, are in accordance with the rheological measurementsshown in FIG. 25A. At 37° C., changes in G′ and θ₂₁₈ values are veryrapid at the early stages of self-assembly, while at 20° C. the rates ofchange of both values are much slower. Therefore, higher storage modulifor gels formed at 37° C. may be attributed to the fast rate of folding.At the initial stages of self-assembly, the high folding rate results inmore nucleation sites for β-sheet rich fibril growth, and, consequently,a network is formed with more junction points. This working mechanism isconsistent with all of the data obtained thus far.

For biomaterials applications it is important to understand the responseof MAX1 to biologically relevant conditions. Therefore, the gelation ofMAX1 was studied in serum free DMEM cell growth media. FIG. 26A shows G′and G″ during the gelation of MAX1 in cell growth media at 37° C.(mammalian cell culturing conditions). At the end of 2 hours of gelationG′ and G″ values were 2300 and 50 Pa, respectively. It can be seen thatthe rate of increase in G′ value at the end of 2 hours is stillsignificant. To eliminate the effects of evaporation that would resultin an increase in peptide concentration and G′ value, the dynamic timesweep experiment was stopped after 2 hours of gelation. (This continuingincrease in the G′ value without showing a final value has also beenseen in gelatin gels). Frequency sweep data, shown in FIG. 26B,indicates that the MAX1-cell growth media solution forms a rigid gelwith G′values ˜2500 Pa. The properties of the frequency sweep data ofthe media induced hydrogel (G′>10×G″, G′ independent of frequency) aresimilar to those shown in FIG. 21A. The response of the hydrogel tosignificant shear was studied by monitoring recovery of G′ afterapplication of high magnitude of strain (1000% at 6 rad/s) to the gelledsolution. Since the applied strain is well outside the linear regime, G′and G″ values are not shown. FIG. 26C shows that after cessation ofshear and the immediate application of 5% strain at 6 rad/s, G′instantaneously recovers almost 50% of its initial value. When recoverydata is compared with the gelation data (FIG. 26A) it can be seen thatthe rate of increase of G′ is faster than the rate during initialgelation. This suggests that during the application of high magnitude ofstrain the physically crosslinked, self-assembled network structure isfractured, resulting in the decrease of connectivity and, thus,elasticity of the material. After the cessation of shear, the networkcan quickly reheal as manifested in the immediate recovery ofsignificant network stiffness. This experiment demonstrates that thesehydrogels are processible and can recover initial rheological propertiesafter being disturbed by external mechanical forces. This ease ofprocessibility and recovery can be advantageous for tissue engineeringapplications (e.g. in vivo injection).

Rigid hydrogels are formed via folding and self-assembly of β-hairpinpeptides. While MAX1 is in a random coil confirmation at physiologicalpH (7.4), the addition of salt to the solution results in the formationof self-assembled structures rich in β-sheet. Rheological datademonstrates that peptide solutions form gels with G′ at least one orderof magnitude greater than G″. In addition, G′ is insensitive tofrequency indicating that the network is similar in elastic propertiesto chemically crosslinked polymer gels. Kinetics of self-assembly aswell as storage modulus of the hydrogels, can be tuned by the ionicstrength of the peptide solution. The network structure is composed ofdense fibrillar assemblies that are crosslinked to each other byphysical junction points possibly due to facial hydrophobic interactionsand hydrogen bonding. The width of the fibrils is approximately 3 nm andthis dimension is in a good agreement with the folded state of thehairpins in the self-assembled state. Salt-triggered self-assembly andconsequent gelation properties are also tuned by temperature. At 37° C.,the kinetics of β-sheet formation and gelation is faster than at 20° C.resulting in stiffer gels. In addition, MAX1 forms rigid and processiblehydrogels in cell growth media at physiological conditions. Thus,properly designed peptides can be triggered by salt to intramolecularlyfold and consequently intermolecularly assemble into supramolecularstructures that result in hydrogels with tunable modulus.

EXAMPLE 2

Encapsulation of Mesenchymal Stem Cell Line C3H10t1/2

To a vial containing 2 mg HPL8 (VKVKVKVKV^(D)PPTKVEVKVKV), 90 μL ofdistilled water at ˜25° C. was added and transferred to an 8 chamberconfocal plate at which time 90 μL of 2×DMEM was added and gentlypipetted to ensure a homogeneous solution optimal for cellencapsulation. Immediately, 20 μL of a 100,000 cells/mL stock solutionin DMEM (serum free) was added to the DMEM/HPL8 mixture and gently mixedto ensure homogeneity, resulting in a 1 wt % HPL8 hydrogel containing20,000 cells. The hydrogel was incubated for 15 minutes at 37° C. and 5%CO₂ to ensure a self-sustaining material. After which time, 400 μL ofDMEM containing 10% FBS was added to the top of the hydrogel allowingfor diffusion of nutrients (media was refreshed after 24 hours).

Live/Dead Assay of Encapsulated C3H10t1/2 Cells

At 48 hours the media was removed from the hydrogel and washed with DMEM(400 μL). A stock solution of 1 μM calcein AM and 2 μM ethidiumhomodimer in DMEM was prepared according to the live/dead assay(Molecular Probes #L3224) package instructions, and 200 μL of this stocksolution was added to the hydrogel. Images of the encapsulated cellswere obtained using a 10× Plan-Apochromat lens on a Zeiss 510 LCMconfocal microscope (488/543 nm) and the results are shown in FIG. 27.Live cells are stained green and dead cells are stained red.

EXAMPLE 3

A peptide-based hydrogelation strategy has been developed that allowshomogenous encapsulation and subsequent delivery of cells, e.g.,C3H10t1/2 mesenchymal stem cells. HPL8, a twenty residue peptide, foldsand self-assembles in response to DMEM cell culture media affordingmechanically rigid hydrogels. Structure-based peptide design was used tomodulate the gelation kinetics of MAX1, the parent peptide, affordingHPL8. When HPL8 hydrogelation is triggered in the presence of cells, thetuned self-assembly kinetics ensures that gels become homogeneouslyimpregnated with cells. A unique characteristic of these constructs isthat when an appropriate shear stress is applied, the gel willshear-thin, becoming a viscous liquid. However, after the application ofshear has stopped, the viscous liquid quickly self-heals producing a gelwith mechanical rigidity nearly identical to the original hydrogel. Thisproperty allows gel/cell constructs to be delivered via syringe withprecision to target sites. Homogenous cellular distribution and cellviability are unaffected by the shear thinning process and gel/cellconstructs stay fixed at the point of introduction suggesting that thesegels may be useful for the delivery of cells to target biological sitesin tissue regeneration efforts.

Hydrogels are heavily hydrated materials finding use in tissueregeneration efforts as extracellular matrix substitutes. For example,preformed hydrogels inserted into cartilage, bone and liver defects inanimal models show potential promise in aiding tissue repair in humans.In addition to preformed gels, “smart” polymeric systems are beingdeveloped that undergo solution-hydrogel phase transitions, in vivo. Inthese systems, either acellular aqueous solutions of polymer orsolutions containing desired cell type(s) are introduced at the tissuesite. Subsequent gelation can occur by taking advantage of environmentaldifferences between the polymeric solution and the in vivo environmentsuch as temperature, ionic strength or enzymatic activity.Alternatively, in vivo gelation can be accomplished by initiating thecrosslinking of photopolymerizing polymer precursors usingcytocompatible photoinitiators. These systems offer the potential ofminimally invasive material implantation by delivering solutions througha catheter inserted into a small incision. Acellular systems result ingels that are designed to be infiltrated by cells from the surroundingtissue, whereas cellular systems afford gel/cell constructs that aredesigned to foster more immediate tissue regeneration. Both systems maycontain growth factors and/or cytokines to enhance tissue regeneration.

Several material properties are commonly studied and often used tobenchmark the potential success of a new material. For example, thecytocompatibility of a material is normally studied by assessingmaterial cytotoxicity, cell adhesion (attachment and subsequentmorphological changes), proliferation, phenotype maintenance, anddifferentiation if progenitor cells are used. Material biocompatibilitymeasures material-induced inflammation and immune response. Also,although not a necessary material attribute, biodegradability can bemeasured. Lastly, the desired bulk mechanical properties such asrigidity, elasticity, compressibility, to name a few, are dependent onthe specific biological application and are a direct consequence of thenano- and microstructure of the hydrogel. For many newly developedhydrogel materials, research mainly focuses on addressing the criteriaoutlined above.

However, for injectable “phase transition” materials, additional andvery important, practical criteria exist which are challenging to meet.Namely, the spatial resolution with which a hydrogel can be introducedin vivo and the ability of the hydrogel to remain localized at the pointof introduction is of paramount importance. A possible severe limitationexists for material systems that are delivered to tissue defects asliquids; unless a well-defined cavity exists that will contain thehydrogel precursor solution, leakage into/onto neighboring tissue isunavoidable and potentially harmful. For bone and cartilage repair, theimplant site can be constrained to limit motion and periosteal flapsused to help spatially restrict material leakage. However, even forwell-defined osteochondral defects, multiple applications of the liquidprecursor may be necessary. For other tissues, well-defined cavities arenot common. In sum, if wide-spread clinical use is anticipated then aninjectable material must be easily administered and stay localized atthe site of introduction.

We have developed a hydrogelation strategy, based on the triggeredself-assembly of peptides, to enable the three-dimensional encapsulationof cells and their subsequent delivery to tissue. The design of thissystem links the intramolecular folding of amphiphilic β-hairpinpeptides to their propensity to self-assemble affording hydrogelmaterial. Peptides are designed such that, when dissolved in aqueoussolutions, they exist in an ensemble of random coil conformationsrendering them fully soluble. However, the addition of an exogenousstimulus results in peptide folding into β-hairpin conformation thatundergoes rapid self-assembly forming a highly crosslinked hydrogel,FIG. 28. Peptides have been designed to fold and assemble in response tochanges in pH or ionic strength, the addition of heat or even light. Inaddition to these stimuli, the addition of cell culture media tobuffered solutions of unfolded peptide triggers folding, self-assemblyand ultimate gelation. FIG. 28 shows this process for the peptide MAX1.Due to electrostatic repulsion between positively charged lysineresidues, MAX1 remains unfolded in low ionic strength buffer at pH 7.4.However, folding can be triggered by screening some of the lysine-basedcharge with the addition of DMEM cell culture media, which containssufficient concentrations of mono- and divalent salts to ensureeffective screening.

In the folded state, MAX1 adopts a hairpin conformation composed of twoβ-strand sequences of alternating hydrophobic and hydrophilic residues(Lys and Val) flanking a tetrapeptide type II′ β-turn. MAX1 hairpins areamphiphilic molecules where one face is hydrophobic and the other faceis hydrophilic. Folded hairpins self-assemble both laterally (via theformation of intermolecular H-bonds and van der Waals contacts) andfacially (via the burial of the hydrophobic face of distinct hairpins),FIG. 28. Detailed structural characterization indicates that MAX1 gelsare comprised of a network of fibrils (10-200 nm) rich in β-sheet. Eachfibril is approximately 3 nm in width, consistent with the folded stateof the molecule. Fibrils are physically crosslinked by non-covalent,hydrophobic interactions between the hydrophobic faces of assembledhairpins and local fibril entanglements. Cryo-TEM and LCSM indicate thatthe gels are well hydrated on both the nano- and micro-length scales,and are microporous. Taken together, these material characteristics areattractive for tissue engineering/regeneration applications. A uniquefeature of these gels is that when an appropriate shear stress isapplied, the gel will shear-thin, becoming a viscous liquid. However,after the application of shear has stopped, the viscous liquid quicklyself-heals producing a gel with mechanical rigidity nearly identical tothe original hydrogel, FIG. 28. Shear thin-recovery processes holdpromise for minimally invasive material delivery.

When hairpin hydrogels are initially formed, gelation can be triggeredin the presence of C3H10t1/2 mesenchymal stem cells affording gels thatare impregnated with cells. Resulting gel/cell constructs can beshear-thin delivered to a targeted secondary site where they quicklyrecover to their original mechanical rigidity with location permanency.In this study, C3H10t1/2 cells are used as a model cell line since theyare sensitive to their environment thus providing a rigorous assessmentof the delivery method with respect to cell viability.

Controlling the rate at which the initial gel is set and the rate atwhich the gel recovers after shear thinning is extremely important. Whengelation is triggered in the presence of cells, the rate of folding andself-assembly must be controlled to ensure homogenous cellincorporation. Gels that form too slowly or quickly would affordnon-homogenous incorporation where cells either sediment to the bottomor are trapped at the top of the container (syringe). Making multipleinjections of non-homogenous gel/cell construct from a single syringeinto different wound sites would result in grossly varied cell densityat each injection site. After injection, gel recovery must be fast toensure that after the viscous solution leaves the syringe and contactsthe tissue, the gel quickly recovers and remains localized at the pointof introduction.

For example, FIG. 29 contains a Laser Scanning Confocal Microscopy(LSCM) cross-sectional view of C3H10t1/2 cells encapsulated within a 0.5wt % MAX1 hydrogel. In this micrograph, cells contained within theinterior of the gel are visualized from the side of the gel (along they-axis, depth of field is 920 μm); the top and bottom of the image isthe top and bottom of the gel, respectively. Cells are encapsulated byfirst dissolving 1.0 wt % MAX1 in buffer at pH 7.4. To this solution, anequal volume (50 μL) of DMEM cell culture medium containing 250,000cells is added. The salt within the DMEM electrostatically screens thelysine side chain charges resulting in peptide folding and consequentself-assembly. This simple one-to-one addition of solutions affords gelsimpregnated with cells. However, it is clear in FIG. 29 that thehydrogelation kinetics of MAX1 are not fast enough to entrap the cellshomogeneously since a majority of the cells sediment to the bottom ofthe gel sample well.

According to our model of folding and self-assembly, the eightpositively charged lysine side chains of MAX1 must be accommodated onthe hydrophilic face for the peptide to fold at pH 7.4. The kinetics ofMAX1 hydrogelation should be hastened by making a point amino acidsubstitution on the hydrophilic face, which lowers the overall chargedensity. By replacing the lysine side chain at position 15 with anegatively charged side chain of glutamic acid, the overall peptidecharge state is lowered by 2. The resultant peptide, HPL8 has a loweramount of positive charge to be screened and should fold and assemblemuch faster than MAX1 in response to identical cell culture conditions.Apart from this, the HPL8 hairpin could possibly be stabilized bycross-strand salt bridge interactions between the glutamic acid andcross-strand lysine residues in the self-assembled state.

FIG. 30 a shows folding and self-assembly kinetics as monitored viacircular dichroism (CD) spectroscopy that shows the onset of meanresidue ellipticity at 216 nm, indicative of β-sheet formation, as afunction of time under solution conditions identical to those used for3-D cell encapsulation. The data clearly show that at 0.5 wt % MAX1requires approximately 30 minutes to completely fold and self-assembleaffording gel while HPL8 is complete in under a minute. This differencein self-assembly kinetics is also clearly observed using oscillatoryrheology (FIG. 30 b) showing the onset of gel rigidity by monitoring thestorage modulus (G′) versus time for 0.5 wt % MAX1 and HPL8 hydrogels.HPL8 forms a rigid hydrogel (G′>100 Pa) within the first 40 seconds andfurther stiffens with time. A G′ of 100 Pa is sufficient to supportcells within the three dimensional confines of the gel network. Incontrast, MAX1 does not reach 40 Pa even after 60 minutes.

This increase in hydrogelation kinetics has little effect on thenanostructure of the HPL8 gel as compared to the MAX1 gel. Transmissionelectron microscopy (TEM) images highlighting the local morphology ofthe fibrils in each gel shows fibril diameters of 3 nm for MAX1 (FIG. 31a) and HPL8 (FIG. 31 b). These dimensions are consistent with the widthof an individual hairpin in the self-assembled state.

Taken together, the data suggests that HPL8 folds and self-assembles ina similar manner to that of MAX1 showing that rational modification ofthe net charge on the hydrophilic face can be used to control thekinetics of β-sheet formation and hydrogelation. Importantly, the LSCMimage of a 0.5 wt % HPL8 hydrogel impregnated with C3H10t1/2 mesenchymalstem cells clearly illustrates that the kinetics of hydrogelation areoptimal for a homogeneous distribution of cells (FIG. 32). In thismicrograph, cells contained within the interior of the gel arevisualized from the side of the gel (along the y-axis, depth of field is920 μm.); the top and bottom of the image is the top and bottom of thegel, respectively.

In order for gel/cell constructs to be delivered by syringe, HPL8 gelsmust exhibit shear thinning behavior, yet recover quickly after deliverywhen they are no longer under shear stress. The ability of acellularHPL8 hydrogel to shear thin and recover was assessed by oscillatoryrheology. The data in FIG. 33 show that after shear thinning in therheometer, the hydrogel immediately recovers. In phase I of thisexperiment, hydrogelation is triggered by the addition of DMEM to a HPL8buffered solution and the onset of G′ is measured as a function of time.A G′>100 Pa is realized within a minute with further stiffening of thenetwork, showing a G′ of 450 Pa at five minutes. In phase II, 1000%strain is applied for 30 seconds to the hydrogel resulting in the shearthinning of the sample and the conversion of the gel to a liquid. Inphase III, the applied strain is decreased to 0.2% and gel recovery ismonitored. The kinetics of recovery are markedly faster than the initialgelation kinetics; recovering gels display a storage modulus of nearly200 Pa within the first 5 seconds of recovery and further stiffen withtime. The fast recovery kinetics are most likely due to the fact thatmany of the non-covalent crosslinks of the gel network remain intactduring shear thinning and only a small fraction needs to re-assemble forthe gel to recover (FIG. 28).

The recovery kinetics are little effected when cells are encapsulated inthe network. Although recovery kinetics of HPL8 gel/cell constructs werenot quantitatively measure by rheology, visually they immediatelyrecovered when shear-thin delivered to a secondary container or surfacevia syringe. Gel/cell constructs can be delivered with high precisionthat is limited only by the gauge of the syringe needle. Recoveredgel/cell constructs are visually clear at these cell-loading densities,self-supporting and remain localized at the point of application evenwhen agitated. The ability of the gel/cell construct to remain localizedwill undoubtedly depend on the surface (tissue) type, but experimentsperformed with tissue culture-treated polystyrene and borosilicatesurfaces as well as human skin demonstrated location permanency (FIG.34). The inset of FIG. 33 b shows a 0.5 wt % HPL8 gel/cell constructthat had been formed directly in a syringe. This construct wassubsequently delivered to a LSCM well and imaged to assess thedistribution of cells within the hydrogel network after shear thinning.FIG. 33 b shows that the cells still retain the homogeneous celldistribution established during initial gel assembly similar to thatshown in FIG. 32.

Lastly, the effect of shear thin delivery on cell viability was assessedby performing a Live-Dead assay on cells that had been encapsulated intoa 0.5 wt % HPL8 hydrogel and subsequently shear thin delivered to a LSCMwell FIG. 33 c. In this micrograph, cells contained within the interiorof the gel are visualized by viewing from the top of the gel (along thez-axis, depth of field is 90 μm.). Three hours after the gel/cellconstruct had been shear-thin delivered, calcien AM and ethidiumhomodimer were added to the top of the gel, which freely diffuse throughthe gel network, staining live cells green and dead cells red,respectively. It is evident that the majority of the cells remain viableafter delivery. In fact, the limited degree of cell death observed inFIG. 33 c is nearly identical to that of the control experiment wherecells were encapsulated in HPL8 hydrogel but not shear thin delivered(FIG. 35).

Results presented herein demonstrate that peptide design can be used togenerate hydrogel materials for specific technological applications.Peptide folding and self-assembly kinetics as well as shear-thinrecovery kinetics have been tuned to allow for the homogenousencapsulation of C3H10t1/2 stem cells within the three dimensionalnetwork of a hydrogel. Resulting gel/cell constructs can be shear thindelivered via syringe to target sites with little effect on thehomogenous distribution of the cells. Cells remain viable during theencapsulation and shear-thin delivery process and gel/cell constructsstay fixed at the point of introduction suggesting that these gels maybe useful for the delivery of cells to target biological sites in tissueregeneration efforts. Long-term cell viability and differentiation ofC3H10t1/2 mesenchymal cells within the hydrogel scaffold are currentlyunder investigation.

Methods

Trifluoroacetic acid (TFA), piperidine, thioanisole, ethanedithiol,N,N-diisopropylethylamine, HEPES, 1,8-Diazabicyclo[5.4.0]undec-7-ene andanisole were purchased from Acros. Appropriately side-chain protectedFmoc-amino acids were purchased from Novabiochem. 1H-Benzotriazolium1-[bis(dimethylamino)methylene]-5chloro-,hexafluoro-phosphate(1-),3-oxide (HCTU) was purchased from Peptides International. 1-methyl2-pyrrolidone, acteonitrile and acetic anhydride were purchased fromFisher. Dulbecco's Modified Eagles Medium and fetal bovine serum werepurchased from Invitrogen. Cell tracker green and Live/Dead assays werepurchased from Molecular Probes. C3H10t1/2 cells were purchased fromATCC.

Peptide Synthesis and Purification

Peptides were synthesized on RINK amide resin via an automated ABI 433Apeptide synthesizer employing standard Fmoc-protocol and HCTUactivation. The resulting dry resin-bound peptides were cleaved andside-chain deprotected for 2 hr under N₂ atmosphere using a TFA:thioanisole: ethanedithiol:anisole (90:5:3:2) cocktail. Filtrationfollowed by ether precipitation afforded crude peptides that werepurified by RP-HPLC (preparative Vydac C 18 peptide/protein column).MAX1: Isocratic at 0% B for 2 min then a linear gradient from 0 to 18% Bover 3 min, then 18 to 100% B over 164 min. Peptide elutes at 29 min. MS(ESI) m/z: 1116.0 [(M+2H)2+, calcd 1115.9]. HPL8. Isocratic at 0% B for2 min then a linear gradient from 0 to 25% B over 17 min, then 25 to 40%B over 30 min, then 40 to 100% B over 10 min. The peptide elutes at 31min. MS (ESI) m/z: 1116.2 [(M+2H)2+, calcd 1116.5]. A flow rate of 8mL/min was employed for preparative HPLC. Elutants for RP-HPLC consistedof solvent A (0.1% TFA in water) and solvent B (90% acetonitrile, 10%water, 0.1% TFA).

Hydrogel Preparation

To a vial containing 1 mg of peptide, 100 μL of 25 mM HEPES, pH 7.4 wasadded resulting in a soluble 1 wt % peptide solution. To this solution,an equal volume of DMEM supplemented with 25 mM HEPES, pH 7.4 was addedto initiate self-assembly, resulting in a 0.5 wt % hydrogel. Allhydrogels were prepared by this method unless otherwise stated.

Circular Dichroism (CD) Experiments

CD kinetic spectra were collected on a Jasco J-810 spectropolarimeteremploying 0.1 mm quartz water-jacketed cell. Peptide samples wereprepared as stated above, transferred to the cell at 37° C. Theellipiticity in millidegrees were monitored at 216 nm as a function oftime. Following the kinetic measurement, a wavelength scan was recordedat 37° C. using a 2 nm step size. The concentrations of MAX1 and HPL8stock solutions were determined by absorbance (λ₂₂₀=15750 cm-1.M-1)after dilution with water. Mean residue ellipticity[θ]=(θ_(obs)/10.1.c)/r, where θ_(obs) is the measured ellipticity inmillidegrees, l is the length of the cell (cm), c is the concentration(M) and r is the number of residues.

Oscillatory Rheology Experiments

Oscillatory rheology experiments were performed on a TA Instrumentsrheometer (AR 2000) with a 25 mm diameter stainless steel parallel plategeometry with a gap height of 0.5 mm. All measurements were acquired at37° C. For dynamic time sweeps (DTS) the hydrogels were prepared asstated above and quickly transferred to the rheometer where the storagemodulus (G′) was monitored as a function of time, frequency=6 rad. sec-1and strain=0.2%. For the shear thinning experiments, a DTS was preformedfor 10 min at which time 1000% stain was applied for 30 seconds to shearthin the material. After which time, the strain was decreased to 0.2%and G′ monitored as a function of time. Dynamic frequency, dynamicstrain and dynamic time sweep measurements measuring both G′ and theloss modulus (G″) are provided in FIG. 36.

Transmission Electron Microscopy Experiments

Hydrogels were prepared as stated above for TEM experiments. A smallvolume of gel (2-5 μL) solution was applied to carbon-coated coppergrids. Samples were negatively stained with 2% (w/v) aqueous uranylacetate. Bright field images of the fibril nanostructure were taken on aJEOL 2000-FX transmission electron microscope at 200 kV acceleratingvoltage on a Gatan CCD camera.

Cell Culture and Confocal Microscopy Experiments

C3H10t1/2 cell growth conditions are 90% DMEM supplemented with 25 mMHEPES, 10% FBS, 5 mM L-glutamine, and 50 μg/mL Gentamicin at 37° C., 5%CO2. For confocal micrographs of hydrogels impregnated with pre-labeledcells, a suspension of 1.5 e6 cells/mL were incubated in a solution of 5μM cell tracker green in DMEM for 45 min. Following labeling, cells werewashed 3× with PBS and re-suspended in DMEM at a concentration of 5 e6cells/mL. An equal volume of the cell suspension was added to a vialcontaining a solution of buffered peptide in 25 mM HEPES (pH 7.4); theresulting solution was immediately transferred to an 8 well borosilicateconfocal plate and placed into the incubator at 37° C., 5% CO2. Forshear thinning experiments the gel/cell constructs were prepared asabove and immediately loaded into a 1 mL syringe equipped with a 20gauge needle and allowed to undergo hydrogelation for 5 min prior toshear thinning onto a confocal plate for imaging. For viability studies,the gel/cell constructs were prepared as above with unlabeled cells.Viability of encapsulated cells in 0.5 wt % hydrogels (before and aftershear thinning) was assessed via a Live/Dead assay at 3 hr afterencapsulation. A stock solution containing both 1 μM calcein AM and 2 μMethidium homodimer in DMEM were prepared according to the Live/Deadassay (Molecular Probes #L3224) package instructions, and 200 μL of thisstock was added to each well. All gel/cell constructs were imaged using10× magnification on a Zeiss 510 LSCM microscope.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims. All patents and publicationscited herein are entirely incorporated herein by reference.

1. A method of making a hydrogel, comprising: providing a solutioncomprising peptides, wherein the peptides comprise a peptide comprisingthe sequence VKVKVKVKV^(D)PPTKVEVKVKV; altering one or morecharacteristics of the solution, wherein a hydrogel is formed.
 2. Amethod according to claim 1, wherein the characteristic is selected froma group consisting of ionic strength, temperature, concentration of aspecific ion, and pH.
 3. A method according to claim 1, wherein alteringcomprises contacting the solution with electromagnetic radiation.
 4. Amethod according to claim 1, wherein altering comprises changing the pHof the solution.
 5. A method according to claim 1, wherein altering oneor more characteristic of the solution results in a salt concentrationof from about 20 mM to about 400 mM.
 6. A method according to claim 5,wherein the salt is NaCl.
 7. A method according to claim 1, wherein thesolution has a pH of less than
 9. 8. A method according to claim 1,wherein the solution has a pH of from about 6.0 to about 8.5.
 9. Amethod according to claim 1, wherein the solution has a pH of from about7.0 to about 8.0.
 10. A method according to claim 1, wherein thesolution has a pH of about 7.4.
 11. A hydrogel, comprising peptideswherein the peptides comprise a peptide comprising the sequenceVKVKVKVKV^(D)PPTKVEVKVKV.
 12. A hydrogel according to claim 11, furthercomprising a salt.
 13. A hydrogel according to claim 12, wherein thesalt is NaCl.
 14. A method of making a hydrogel, comprising: injecting asolution comprising peptides into an animal, wherein the solution formsa hydrogel inside the animal and wherein the peptides comprise a peptidecomprising the sequence VKVKVKVKV^(D)PPTKVEVKVKV.
 15. A method accordingto claim 14, wherein the animal is a mammal.
 16. A method according toclaim 14, wherein the animal is a human.
 17. A method according to claim14, wherein the solution further comprises one or more therapeuticagent.
 18. A method according to claim 17, wherein the therapeutic agentis selected from a group consisting of small molecules, peptides,proteins, and cells.
 19. A method of delivering a therapeutic agent toan animal in need thereof, comprising: administering a solutioncomprising the therapeutic agent and one or more peptides to the animal,wherein the solution forms a hydrogel inside the animal and wherein thepeptides comprise a peptide comprising the sequenceVKVKVKVKV^(D)PPTKVEVKVKV.
 20. A method according to claim 19, whereinthe animal is a mammal.
 21. A method according to claim 19, wherein theanimal is a human.
 22. A method according to claim 19, wherein thetherapeutic agent is selected from a group consisting of smallmolecules, peptides, proteins, and cells.
 23. A method of delivering atherapeutic agent to an animal in need thereof, comprising:administering a hydrogel comprising the therapeutic agent and one ormore peptides to the animal, wherein the peptides comprise a peptidecomprising the sequence VKVKVKVKV^(D)PPTKVEVKVKV.
 24. A method accordingto claim 23, wherein the animal is a mammal.
 25. A method according toclaim 23, wherein the animal is a human.
 26. A method according to claim23, wherein the therapeutic agent is selected from a group consisting ofsmall molecules, peptides, proteins, and cells.
 27. A method of treatinga wound in an animal, comprising: contacting the wound with a solutioncomprising a peptide, wherein the solution forms a hydrogel and whereinthe peptide comprises the sequence VKVKVKVKV^(D)PPTKVEVKVKV.
 28. Amethod according to claim 27, wherein the solution comprises atherapeutic agent.
 29. A method according to claim 27, wherein theanimal is a mammal.
 30. A method according to claim 27, wherein theanimal is a human.
 31. A method according to claim 28, wherein thetherapeutic agent is selected from a group consisting of smallmolecules, peptides, proteins, and cells.
 32. A method of growing cells,comprising: forming a hydrogel comprising cells, wherein the hydrogelcomprises a peptide comprising the sequence VKVKVKVKV^(D)PPTKVEVKVKV;and maintaining the cells under conditions suitable for cell viability.33. A method according to claim 32, wherein forming a hydrogel comprisesadjusting one or more characteristic of a solution comprising peptides.34. A method according to claim 33, wherein the characteristic adjustedis selected from a group consisting of pH, ionic strength, and specificion concentration.
 35. A method according to claim 33, wherein thecharacteristic adjusted is ionic strength.
 36. A method according toclaim 33, wherein the characteristic adjusted is Ca²⁺ ion concentration.37. A method according to claim 32, wherein the cells are animal cells.38. A method according to claim 32, wherein the cells are mammaliancells.
 39. A method according to claim 32, wherein the cells are humancells.
 40. A method according to claim 32, wherein the cells areosteoblasts.
 41. A method according to claim 32, wherein the cells arefibroblasts.
 42. A method according to claim 32, wherein the cells arerecombinant.
 43. A method according to claim 32, wherein the cellsexpress a protein of interest.
 44. A method according to claim 44,wherein the protein of interest is an antibody.
 45. A method accordingto claim 32, wherein the cells are stem cells.
 46. A sensor comprising ahydrogel, wherein the hydrogel comprises a peptide comprising thesequence VKVKVKVKV^(D)PPTKVEVKVKV.
 47. A sensor according to claim 46,wherein one or more characteristic of the hydrogel is altered when thehydrogel is contacted with an analyte of interest.
 48. A sensoraccording to claim 47, wherein the characteristic altered is stiffness.49. A sensor according to claim 47, wherein the characteristic alteredis an optical property.
 50. A method of detecting environmentalconditions, comprising: contacting a sensor comprising a hydrogel with asample representative of the environmental conditions; and determiningone or more characteristic of the hydrogel, wherein the hydrogelcomprises a peptide comprising the sequence VKVKVKVKV^(D)PPTKVEVKVKV.51. A method according to claim 50, wherein one or more characteristicof the hydrogel is altered when the hydrogel is contacted with ananalyte of interest.
 52. A method according to claim 51, wherein thecharacteristic altered is stiffness.
 53. A method according to claim 51,wherein the characteristic altered is an optical property.
 54. A methodaccording to claim 50, further comprising comparing the characteristicof the hydrogel to the same characteristic of the hydrogel determined ata different time.
 55. A method of purifying a molecule of interest,comprising: contacting a solution comprising the molecule of interestand one or more contaminants with a hydrogel under conditions causingthe molecule of interest to be retained by the hydrogel; and recoveringthe molecule of interest from the hydrogel, wherein the hydrogelcomprise a peptide comprising the sequence VKVKVKVKV^(D)PPTKVEVKVKV. 56.A method according to claim 55, wherein the molecule of interest is aprotein.
 57. A method according to claim 55, wherein the molecule ofinterest is an antibody.
 58. A method according to claim 55, wherein themolecule of interest is a therapeutic agent.
 59. A method of purifying amolecule of interest, comprising: contacting a solution comprising themolecule of interest and one or more contaminants with a hydrogel underconditions causing at least one contaminant to be retained by thehydrogel; and recovering the molecule of interest, wherein the hydrogelcomprise a peptide comprising the sequence VKVKVKVKV^(D)PPTKVEVKVKV. 60.A method according to claim 59, wherein the molecule of interest is aprotein.
 61. A method according to claim 59, wherein the molecule ofinterest is an antibody.
 62. A method according to claim 59, wherein themolecule of interest is a therapeutic agent.
 63. A method ofencapsulating cells in a hydrogel, comprising: providing a solutioncomprising peptides and a solution comprising cells, wherein thepeptides comprise a peptide comprising the sequenceVKVKVKVKV^(D)PPTKVEVKVKV; combining the solutions under conditionswherein a characteristic of the solution comprising peptides is alteredsuch that a hydrogel is formed.
 64. A method according to claim 63,wherein the characteristic adjusted is selected from a group consistingof pH, ionic strength, and specific ion concentration.
 65. A methodaccording to claim 63, wherein the characteristic adjusted is ionicstrength.
 66. A method according to claim 63, wherein the characteristicadjusted is Ca²⁺ ion concentration.
 67. A method according to claim 63,wherein the cells are animal cells.
 68. A method according to claim 63,wherein the cells are mammalian cells.
 69. A method according to claim63, wherein the cells are human cells.
 70. A method according to claim63, wherein the cells are osteoblasts.
 71. A method according to claim63, wherein the cells are fibroblasts.
 72. A method according to claim63, wherein the cells are recombinant.
 73. A method according to claim63, wherein the cells express a protein of interest.
 74. A methodaccording to claim 73, wherein the protein of interest is an antibody.75. A method according to claim 63, wherein the cells are stem cells.76. A method according to claim 63, wherein the cells are human stemcells.
 77. A hydrogel comprising peptides and cells, wherein the cellsare evenly distributed throughout the hydrogel.
 78. A hydrogel accordingto claim 77, wherein the peptides comprise a peptide comprising thesequence VKVKVKVKV^(D)PPTKVEVKVKV is HPL8 (VKVKVKVKV^(D)PPTKVEVKVKV)(SEQ ID NO:21).
 79. A hydrogel according to claim 77, wherein the cellsare animal cells.
 80. A hydrogel according to claim 77, wherein thecells are mammalian cells.
 81. A hydrogel according to claim 77, whereinthe cells are human cells.
 82. A hydrogel according to claim 77, whereinthe cells are osteoblasts.
 83. A hydrogel according to claim 77, whereinthe cells are fibroblasts.
 84. A hydrogel according to claim 77, whereinthe cells are recombinant.
 85. A hydrogel according to claim 77, whereinthe cells express a protein of interest.
 86. A hydrogel according toclaim 85, wherein the protein of interest is an antibody.
 87. A hydrogelaccording to claim 77, wherein the cells are stem cells.
 88. A hydrogelaccording to claim 77, wherein the cells are human stem cells.